MEDICAL    SCHOOL 


Gift  of 
Dr.   Ernest  L.  Walker 


AN  INTRODUCTION 


TO   THE    STUDY   OF 


INFECTION  AND  IMMUNITY 


INCLUDING   CHAPTERS   ON 


SERUM  THERAPY,  VACCINE  THERAPY,  CHEMOTHERAPY 
AND  SERUM  DIAGNOSIS 


FOE  STUDENTS  AND  PRACTITIONERS 


CHARLES  E.  SIMON,  B.A.,  M.D. 

PROFESSOR  OF  CLINICAL  PATHOLOGY  AND  EXPERIMENTAL  MEDICINE  AT  THE  COLLEGE  OF  PHYSICIANS  AND 
SURGEONS;  PATHOLOGIST  OF  THE  UNION  PROTESTANT  INFIRMARY  AND  THE  HOSPITAL 

FOR  THE   WOMEN  OF  MARYLAND;    CLINICAL  PATHOLOGIST  TO  THE 
MERCY  HOSPITAL  OF  BALTIMORE,   MARYLAND 


SECOND  EDITION,  REVISED  AND  ENLARGED 


ILLUSTRATED 


LEA   &   FEBIGER 

PHILADELPHIA    AND    NEW    YORK 


Entered  according  to  the  Act  of  Congress,  in  the  year  1913,  by 

LEA  &  FEBIGER, 
in  the  office  of  the  Librarian  of  Congress.     All  rights  reserved. 


TO 
PAUL    EHRLICH 

THE    GRANDMASTER    OF    EXPERIMENTAL    MEDICINE 

THIS    VOLUME 

IS   RESPECTFULLY  DEDICATED 
BY 

THE    AUTHOR 


*\  ^  9  <  i  W 

0  4  fv  «  '  O 


PREFACE  TO  SECOND  EDITION 


THE  fact  that  the  first  edition  of  Infection  and  Immunity  has 
been  exhausted  within  a  year  indicates  the  interest  with  which 
the  general  practitioner  is  meeting  the  marvellous  advances  of 
modern  experimental  medicine.  In  preparing  this  new  edition  the 
author  has  endeavored  to  embody  the  most  notable  achievements 
of  the  past  year,  and  has  thus  added  sections  on  auto-  and  normal 
serum  therapy,  on  the  chemotherapy  of  pneumococcus  infections 
and  of  cancer,  on  the  serum  diagnosis  of  pregnancy,  etc.,  besides 
giving  the  entire  work  a  careful  review. 

The  author  hopes  that  his  work  has  served  to  some  extent  in 
diffusing  a  knowledge  of  this  vastly  important  subject,  and  in 
making  it  readily  assimilable  and  applicable,  and  he  hopes  that 
this  new  and  revised  edition  may  meet  with  the  same  generous 

recognition  as  its  predecessor. 

C.  E.  S. 


CONTENTS 


CHAPTER   I 
INTRODUCTION 

Immunology 19 

Infection  and  Infectious  Diseases 20 


CHAPTER   II 
THE  NATURE  OF  INFECTION 

Local  Conditions  and  Infection 23 

Obstacles  to  Infection 25 

CHAPTER  III 

THE  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

Classification  of  Microparasites 

Virulence  and  Aggressivity 32 

Aggressins 35 

Passive  Aggressivity 35 

Active  Aggressivity 41 

CHAPTER   IV 

BACTERIAL  POISONS 

Ptomains 48 

Toxins 49 

Endotoxins 52 

Bacterial  Proteins .52 

Infection  with  Animal  Parasites ....       55 

CHAPTER  V 

THE  DEFENSIVE  FORCES  OF  THE  MICROORGANISM 

Phagocytosis 57 

The  Opsonins  and  Bacteriotropins .61 

Chemotaxis  65 


vni  CONTENTS 

CHAPTER  VI 

THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

The  Alexins 69 

Pfeiffer's  Experiment 71 

The  Leukins 79 

Offensive-defensive  Mechanism  in  Infections  with  Necroparasites    ...  80 

Offensive-defensive  Mechanism  in  Infections  with  True  Parasites    .      .      .  81 

Offensive-defensive  Mechanism  in  Infections  with  Semiparasites      ...  85 

CHAPTER  VII 
ANTIGENS  AND  ANTIBODIES 

Allergia 91 

Antitoxins 92 

Bacteriolysins 94 

Agglutinins 95 

Precipitins 96 

Cytolysins 97 

Isocytolysins 100 

Auto-antibodies 100 

Immune  Opsonins  and  Bacteriotropins 101 

Antiferments 102 

Antilipoids ' 103 

Albuminolysins 103 

Anaphylaxis 104 

CHAPTER  VIII 

THE  SIDE-CHAIN  THEORY 

Formation  of  Antibodies 114 

CHAPTER   IX 
THE  DIFFERENT  TYPES  OF  IMMUNITY 

Natural  Immunity 127 

Acquired  Immunity 131 

Antitoxic  Immunity 132 

Athreptic  Immunity 134 

Antiaggressin  Immunity 135 

Antibacterial  Immunity 137 

CHAPTER  X 
ANAPHYLAXIS 

Richet's  Studies 146 

The  Phenomenon  of  Arthus    .  147 


CONTENTS  ix 

Studies  of  v.  Pirquet 147 

The  Phenomenon  of  Theobald  Smith      .            148 

Antianaphylaxis 149 

Serum  Sickness 150 

Passive  Anaphylaxio 150 

The  Anaphylactic  Shock 151 

The  Anaphylactic  Toxin 152 


CHAPTER  XI 
ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 

Anaphylaxis  in  its  Relation  to  Disease 158 

The  Idiosyncrasies  and  Anaphylaxis 167 

CHAPTER  XII 
ACTIVE  IMMUNIZATION 

(A)  For  Prophylactic  Purposes 174 

Against  Smallpox .  175 

Against  Rabies 183 

Against  Typhoid  Fever 189 

Against  Cholera 195 

Against  Plague 197 

Against  Dysentery 200 

(B)  For  Therapeutic  Purposes 200 

In  Pyogenic  Infections 205 

In  Tuberculosis 206 

Estimation  of  the  Opsonic  Content  of  the  Blood    ....  214 


CHAPTER  XIII 

PASSIVE  IMMUNIZATION 

Antitoxic  Immunization 222 

Against  Diphtheria 223 

Against  Tetanus 234 

Against  Dysentery 238 

Against  Cholera 240 

Against  Typhoid  Fever 241 

Against  Plague 241 

Bacteriolytic-bacteriotropic  Immunization 241 

Against  Meningococcus  Meningitis 242 

Against  Streptococcus  Infections 246 

Against  Staphylococcus  and  Pneumococcus  Infections      ....  252 

Against  Gonococcus  Infections 254 

Autoserum  Therapy 255 

Normal  Serum  Therapy .  257 


x  CONTENTS 

CHAPTER  XIV 
CHEMOTHERAPY 

Chemotherapy 259 

Salvarsan  and  its  Use  in  Syphilis 265 

Neosalvarsan 268 

Salvarsan  and  its  Use  in  Non-syphilitic  Maladies 276 

Chemotherapy  in  Bacterial  Infections 277 

Chemotherapy  in  Malignant  Disease 278 

CHAPTER  XV 
THE  APPLICATION  OF  IMMUNOLOGICAL  PRINCIPLES  TO  DIAGNOSIS 

The  Agglutination  Reactions 282 

The  Widal  Reaction 284 

Bacteriolytic  Reactions 288 

Diagnostic  Reactions  Depending  upon  Complement  Fixation  .  .  .  290 

The  Wassermann  Reaction 291 

Precipitin  Reactions 304 

The  Biological  Blood  Test 305 

Ferment  Reactions 307 

Abderhalden's  Pregnancy  Test .  307 

Allergic  Reactions 310 

The  Tuberculin  Test 311 

The  Luetin  Reaction  .  317 


INFECTION  AND  IMMUNITY 


CHAPTER    I 
INTRODUCTION 

A  SURVEY  of  the  earliest  writings  in  medicine  shows  that  many  if 
not  all  the  diseases  with  which  we  have  to  deal  today  were  existing 
then.  Smallpox,  plague,  cholera,  typhoid  fever,  dysentery,  diph- 
theria, tuberculosis,  malaria,  erysipelas,  measles,  scarlatina,  rabies 
were  known  to  Hippocrates  and  Galen  as  they  are  to  us;  wound 
infections  existed  then  as  now;  nephritis,  diabetes,  rheumatism,  gout, 
various  types  of  anemia,  cancer,  etc.,  occupied  the  physicians  of 
the  days  of  the  Pharaohs  as  they  do  those  of  the  present  century. 
This  acknowledgment  carries  with  it  the  admission  that  during 
all  these  centuries  the  physician  has  not  been  able  to  master  these 
diseases. 

Much  progress  has  of  course  been  made,  but  much  more  still 
remains  to  be  done.  This  is  no  reflection  upon  the  medical  men  of 
the  past;  they  have  accomplished  their  share  in  the  evolution  of 
medical  science,  and  it  is  unnecessary  to  point  out  at  this  place 
how  well  this  has  been  done.  Since  medical  science  depends  for  its 
own  progress  upon  progress  in  the  subservient  sciences,  the  rapid 
evolution  of  modern  medicine  is  the  direct  result  of  the  wonderful 
advances  in  the  domain  of  chemistry,  physics,  and  the  various 
branches  of  biology.  In  the  dark  days  of  the  medieval  ages  active 
progress  was  out  of  the  question,  and  it  is  no  wonder  that  therapeutic 
empiricism  sank  to  its  lowest  level.  Material  advance  of  medical 
science  as  a  science  could  only  be  possible  after  a  foundation  had 
been  created,  of  which  anatomy,  physiology,  pathology,  bacteriology, 
and  modern  pharmacology  are  integral  components,  and  as  the 
latter  four  are  the  product  of  the  last  century  almost  exclusively, 
nay  even  of  the  last  fifty  years,  there  is  small  cause  for  wonder  that 
2 


18  INTRODUCTION 

so  little  has  been  accomplished  during  the  many  centuries  that  have 
passed,  and  at  the  same  time  that  so  much  has  been  achieved  in 
the  brief  period  that  has  really  been  available  for  productive 
work. 

The  days  of  therapeutic  empiricism  are  fortunately  coming  to  an 
end.  From  the  standpoint  of  curative  therapy  they  have  brought 
us  but  little  that  is  worth  retaining — cinchona  bark,  the  gift  of  the 
Peruvian  Indian,  for  the  treatment  of  malaria,  and  mercury,  a  remedy 
of  the  Talmists,  as  a  problematical  cure  for  syphilis.  As  regards  the 
curative  treatment  of  the  remainder,  not  one  of  the  hundreds  and 
thousands  of  pharmaceutical  preparations  that  have  been  intro- 
duced since  the  days  of  the  Vedas  has  been  shown  to  be  of  value, 
if  as  evidence  of  a  curative  effect  we  demand  a  shortening  of  that 
period  of  time  which  the  animal  body  itself  requires  to  accomplish 
a  cure.  We  have  learned  to  prevent  many  diseases  by  the  elimina- 
tion of  the  corresponding  infecting  agents  from  our  midst;  cholera, 
plague,  typhus  fever,  typhoid  fever,  yellow  fever,  smallpox,  malaria, 
and  diphtheria  are  diseases  which  if  they  still  exist  among  civilized 
people  do  so  with  the  consent  of  the  people  in  the  face  of  a  full 
knowledge  of  the  manner  of  their  prevention. 

Wonderful  progress  also  has  been  made  in  surgery.  By  its  means 
countless  lives  have  been  saved  which  otherwise  wxmld  have  been 
doomed.  But  after  all  surgical  treatment  cannot  be  regarded  as 
curative  treatment  in  the  proper  sense  of  the  word;  the  surgeon  may 
amputate  a  badly  crushed  limb  or  he  may  remove  a  diseased  appen- 
dix, or  a  cancerous  breast,  but  he  does  not  cure  the  limb,  nor  the 
appendix,  nor  does  he  restore  the  breast  to  its  original  condition. 
The  final  repair,  the  healing  of  the  wound,  is  accomplished  by  the 
animal  body  itself.  The  surgeon,  however,  is  frequently  placed  in  a 
condition  where  he  can  assist  nature  materially  to  accomplish 
a  cure,  and  in  this  respect  he  is  certainly  more  favorably  placed 
than  the  internist. 

The  latter  may  be  a  most  skilful  diagnostician,  an  excellent 
pathologist  perhaps,  but  he  does  not  cure  the  diseases  with  which 
he  is  brought  into  contact.  He  may  in  a  measure  influence  some  dis- 
eases by  his  directions  for  the  general  care  of  the  patient,  but,  as  a  rule, 
the  patient  dies  or  recovers  irrespective  of  his  therapeutic  efforts, 
insofar  at  least  as  these  efforts  are  based  upon  ancient  empiricism. 
Typhoid  fever  patients  still  pursue  the  same  course  which  was  so  well 


IMMUNOLOGY  19 

described  by  the  physicians  of  the  medieval  ages;  our  pneumonia 
death-rate  is  still  what  it  was  when  the  earliest  records  on  the  subject 
were  kept,  and  is  virtually  the  same  for  the  millionaire  in  his  marble 
palace,  surrounded  by  doctors  and  nurses,  as  for  the  tramp  who  is 
cared  for  by  the  roadside  by  his  brother  tramps.  The  "virulence"  of 
an  epidemic  of  scarlatina  or  measles  may  vary,  but  our  death-rate  in 
the  long  run  is  virtually  the  same.  Where  actual  progress  has  been 
made  in  the  treatment  of  disease,  such  progress  has  been  due  not  to 
our  therapeutic  interference  by  means  of  drugs,  but  to  a  recognition, 
be  it  ever  so  slight,  of  those  factors  by  wrhich  nature  herself,  unaided 
and  at  the  same  time  unhampered  by  empirical  drug  treatment,  seeks 
to  accomplish  that  end.  For  after  all  the  very  thing  which  physicians 
have  sought  to  accomplish  in  all  the  centuries  that  have  passed,  viz., 
the  cure  of  disease,  that  very  thing  nature  has  accomplished  by 
herself,  before  our  very  eyes,  countless  millions  of  times. 

Nature  herself  cures  75  per  cent,  of  the  pneumonia  cases,  while 
the  physician  fails  to  cure  any,  for  surely  he  cannot  claim  as  his  own 
what  nature  does,  and  he  evidently  loses  the  25  per  cent,  that  nature 
loses.  The  fact  that  nature  does  not  cure  all  cases  could  of  course 
be  interpreted  as  indicating  that  the  means  at  nature's  command 
are  after  all  not  perfect.  That  is  naturally  a  debatable  point.  So 
much,  however,  seems  certain  that  nature's  ways,  so  far  as  we  have 
become  familiar  with  them,  are  the  only  specific  ways  along  which 
progress  seems  possible,  and  that  drug  treatment,  if  it  ever  shall 
become  of  value,  must  start  from  a  different  basis,  and  that  basis 
must  be  a  knowledge  of  the  principles  which  underlie  the  interaction 
between  the  disease-producing  agent  and  the  affected  organism. 

Immunology. — The  study  of  these  forces  constitutes  the  domain  of 
immunology,  of  which  in  turn  modern  chemotherapy,  serum  therapy, 
and  vaccine  therapy  are  the  logical  products.  The  earliest  work  in 
this  direction  is  intimately  linked  with  the  name  of  Pasteur,  and 
constitutes  the  basis  of  all  future  wrork.  It  was  Pasteur  who  first 
demonstrated  that  material  progress  in  the  treatment  and  prevention 
of  the  so-called  infectious  diseases  could  only  be  achieved  by  the 
recognition  of  the  fact  that  the  production  of  active  resistance  to 
an  infecting  agent  on  the  part  of  a  susceptible  animal  necessitates 
the  introduction  of  the  infecting  agent  into  the  animal  body;  in 
other  words,  that  acquired  immunity,  be  this  absolute  or  relative, 
temporary  or  permanent,  is  merely  a  phase  of  infection.  The  study 


20  INTRODUCTION 

of  infection  then  may  be  regarded  as  the  key-note  to  the  entire 
problem  of  the  infectious  diseases.  How  does  infection  primarily 
take  place?  how  does  infection  give  rise  to  disease?  and  how  does 
the  animal  body  overcome  infection?  these  are  the  most  important 
questions  which  at  present  occupy  the  attention  of  immunologists 
the  world  over,  and  it  is  the  object  of  the  present  work  to  present 
to  the  practising  physician  the  more  important  data  which  have 
already  been  worked  out. 

Infection  and  Infectious  Disease. — In  the  earliest  days  of  bacteri- 
ology, when  the  pathogenic  role  of  various  bacteria  was  just  be- 
ginning to  be  understood,  it  was  thought  that  the  presence  of  such 
organisms  in  the  animal  body  could  only  be  anticipated  if  symptoms 
of  the  corresponding  disease  existed  at  the  same  time,  or  were  about 
to  appear;  in  other  words,  the  presence  of  a  pathogenic  bacterium 
in  the  body  of  an  individual  was  looked'  upon  as  equivalent  to  in- 
fection, and  the  terms  infection  and  infectious  disease  were  prac- 
tically used  synonymously.  This  conception  of  the  terms  seemed 
quite  warrantable  at  the  time  in  view  of  the  findings  in  such  a  dis- 
ease as  tuberculosis,  where  it  had  just  been  established  that  the 
disease  in  question  was  invariably  associated  with  the  presence  of 
the  tubercle  bacillus,  while  the  existence  of  the  tubercle  bacillus  in 
the  body  in  the  absence  of  a  corresponding  lesion  was  unknown. 
The  majority  of  physicians  hence  readily  accepted  this  line  of  thought, 
which  future  investigations  have  shown  to  be  erroneous.  For  it 
was  soon  demonstrated  that  pathogenic  organisms  may  be  present 
on  the  tegumentary  and  mucous  surfaces  of  the  body  without  con- 
comitant disease. 

It  is  thus  well  known  that  staphylococci  are  present  at  almost 
any  point  of  the  skin,  and  that  streptococci  even  may  here  be  demon- 
strated in  perfectly  healthy  individuals.  Pneumococci  may  be 
found  in  the  mouths  of  almost  every  individual,  non-virulent  to  be 
sure,  in  the  majority  of  people,  but  virulent  in  fully  15  to  20  per 
cent,  of  the  cases,  in  the  absence  of  any  symptoms  of  disease.  Strep- 
tococci are  here  likewise  not  infrequent.  Tubercle  bacilli  have  been 
found  in  the  nasal  secretion  of  healthy  attendants  on  tubercular 
patients.  Diphtheria  bacilli  are  frequently  encountered  in  those 
who  have  been  about  diphtheria  patients,  and  normal  carriers  of 
the  meningococcus,  in  districts  in  which  the  corresponding  disease 
is  prevalent,  are  frequently  more  common  than  patients  with  the 


INFECTION  AND  INFECTIOUS  DISEASE  21 

disease.  Then,  in  the  normal  intestinal  contents  there  are  myriads 
of  bacteria,  the  majority  of  them  harmless  saprophytes,  it  is  true, 
but  in  addition  there  are  also  staphylococci,  streptococci,  colon 
bacilli,  and  in  herbivorous  animals  the  tetanus  bacillus  and  the 
anthrax  bacillus. 

Evidently,  then,  the  mere  presence  of  the  disease-producing 
organisms  on  the  tegumentary  and  mucous  surfaces  of  the  body 
does  not  indicate  either  that  the  individual  has  passed  through  the 
disease  in  question,  or  is  ill  at  the  time,  or  is  about  to  fall  ill;  nor 
does  the  mere  presence  of  such  organisms  constitute  infection.  If, 
however,  the  normal  epithelial  barrier  has  once  been  passed  and  the 
deeper  structures  have  been  invaded  then  we  can  speak  of  infection, 
and  wrhen  infection  has  once  taken  place  then  we  may  also  find 
clinical  evidence  of  such  infection,  i.  e.,  symptoms  of  the  corre- 
sponding infectious  disease;  but  it  does  not  follow  because  infection 
has  taken  place  that  symptoms  of  disease  must  of  necessity 
develop. 

Infection  and  infectious  disease  are  thus  not  synonymous  terms. 
The  two  may  be  associated,  but  they  are  not  necessarily  so.  Infec- 
tion probably  always  results  in  a  disturbance  of  the  normal  functions 
of  the  host,  and  if  this  disturbance  rises  beyond  a  certain  point, 
symptoms  may  develop  which  constitute  what  clinicians  regard 
as  the  corresponding  infectious  disease.  If,  however,  the  normal 
functional  equilibrium  of  the  host  is  but  little  affected  by  the  pres- 
ence of  the  invading  organism,  no  clinical  symptoms  of  disease 
develop,  notwithstanding  the  fact  that  the  microorganisms  may 
have  multiplied  in  the  body  to  an  enormous  extent.  There  would 
thus  be  an  infection,  but  no  infectious  disease,  using  the  term  disease 
in  the  ordinary  sense  of  the  word.  In  some  cases  of  this  kind,  as  in 
anthrax  in  sheep,  for  example,  the  infection  may  nevertheless  result 
fatally,  but  the  period  of  time  during  which  the  animal  shows  clinical 
symptoms  of  infection  is  so  brief  that  one  can  hardly  speak  of  evi- 
dence of  disease;  when  this  appears  death  is  virtually  at  hand.  In 
other  cases,  such  as  some  of  the  protozoan  infections  of  the  blood  of 
various  animals  (ordinary  rat  trypanosomiasis,  for  example),  no 
harm  seems  to  result  to  the  host  whatever,  even  though  the  blood 
be  swarming  with  the  invaders.  The  same  may  at  times  be  noted  in 
partially  immunized  animals,  when  extensive  infection  of  the  peri- 
toneal cavity  may  be  produced  in  the  case  of  such  organisms,  as  the 


22  INTRODUCTION 

anthrax  and  the  swine  plague  bacillus,  without  any  concomitant 
evidence  of  disturbance  of  the  host.  There  may  thus  be  infection 
of  a  high  order  without  any  evidence  of  associated  disease. 

Evidently,  then,  it  is  necessary  to  distinguish  sharply  between 
(a)  mere  surface  invasions,  (6)  infection  proper,  and  (c)  infectious 
disease.  The  first  subject  belongs  essentially  to  the  epidemiologist 
and  the  sanitarian,  while  the  study  of  infection  and  infectious  dis- 
ease engage  the  attention  of  the  immunologist. 


CHAPTER  II 
THE  NATURE  OF  INFECTION 

IN  studying  the  subject  of  infection,  one  of  the  first  questions 
which  naturally  suggests  itself  is:  Why  does  infection  not  always 
follow  primary  invasion?  In  some  cases  it  might  be  argued  that 
the  invasion  at  the  time  of  observation  was  not  primary,  and  that 
the  person  in  question  may  have  acquired  immunity  to  the  micro- 
organism under  consideration  at  an  earlier  date,  but  that  the  infec- 
tion at  the  time  was  so  mild  as  to  have  escaped  detection.  In  the 
case  of  such  organisms  as  the  typhoid  bacillus,  the  plague  bacillus, 
and  the  cholera  bacillus,  such  an  explanation  might  be  warrantable 
in  some  cases;  but  it  is  evidently  an  explanation  for  which  proof 
would  be  difficult,  if  not  impossible  to  furnish;  it  would  be  a  mere 
assumption  without  any  adequate  basis. 

Then,  again,  it  might  be  argued  that  infection  does  not  occur 
owing  to  the  existence  of  a  natural  general  immunity;  but,  as  a  matter 
of  fact,  it  is  extremely  doubtful  on  the  one  hand  whether  an  abso- 
lute natural  immunity  really  exists  among  individuals  of  a  species 
which  is  known  to  be  generally  susceptible  to  infection  with  a  given 
organism,  and,  on  the  other  hand,  we  find  that  some  individuals 
actually  do  become  infected  at  a  later  date,  showing  that  they  were 
in  reality  not  immune.  With  such  organisms,  moreover,  as  the 
pneumococcus,  influenza  bacillus,  staphylococcus,  streptococcus,  and 
diphtheria  bacillus,  infection  even  does  not  give  rise  to  an  immunity 
that  is  deserving  of  the  name;  on  the  contrary  it  leads  to  hyper- 
sensitiveness  and  not  to  increased  resistance.  We  can  accordingly 
discard  the  assumption  that  a  general  immunity  is  an  important 
factor  in  discussing  the  reasons  why  primary  invasion  does  not 
always  lead  to  actual  infection. 

Local  Conditions  and  Infection. — On  the  other  hand  it  is  conceivable 
that  local  conditions  may  exist  which  would  prevent  the  penetra- 
tion of  a  microorganism  into  the  deeper  tissues  from  certain  surfaces, 
while  from  others  this  would  be  possible.  As  a  matter  of  fact  there 


24  THE  NATURE  OF  INFECTION 

is  a  good  deal  of  evidence  to  show  that  local  conditions  are  of  the 
greatest  importance  in  determining  the  occurrence  or  non-occur- 
rence of  infection.  It  is  thus  well  known  that  infection  with  the 
cholera  vibrio,  the  typhoid  or  the  dysentery  bacillus  can  only  occur 
from  the  digestive  tract,  while  the  gonococcus  shows  a  marked  pre- 
dilection for  the  genital  tract  and  the  conjunctiva,  and  the  meningo- 
coccus  for  the  upper  respiratory  tract.  The  staphylococcus  and 
streptococcus,  on  the  other  hand,  as  well  as  the  plague  bacillus  may 
infect  from  almost  any  point,  and  the  same  probably  is  true  of  the 
pneumococcus,  although  its  special  affinity  is  directed  to  the  respira- 
tory tract.  It  might  be  argued,  of  course,  that  the  organisms  in 
question  do  not  meet  with  more  favorable  conditions  for  infection 
at  the  points  where  this  usually  occurs  than  would  be  the  case 
elsewhere,  and  that  they  infect  from  these  points  largely  because 
they  are  the  only  regions  which  are  usually  open  to  invasion.  There 
is  no  good  evidence,  however,  to  support  such  a  claim,  while  a  num- 
ber of  data  go  to  show  that  there  are  unquestionably  definite  dis- 
tricts which  are  more  prone  to  become  points  of  infection  with  specific 
organisms  than  others,  because  of  purely  local  conditions.  The 
gonococcus  and  diphtheria  bacillus  are  thus  incapable  of  producing 
an  infection  through  the  skin,  even  when  this  has  been  previously 
wounded  at  the  point  of  contact  with  the  organisms  in  question. 
The  cholera  vibrio  can  infect  only  from  the  intestinal  mucosa, 
but  not  from  the  mouth,  the  esophagus,  the  stomach,  or  the  genital 
tract.  In  the  stomach,  indeed,  an  active  multiplication  of  the 
organism  in  question  cannot  occur,  as  the  cholera  vibrio  is  rapidly 
destroyed  by  the  hydrochloric  acid  of  the  gastric  juice. 

While  local  conditions  are  thus  unquestionably  of  moment  in 
determining  a  liability  to  infection  and  primary  invasion  on  the  part 
of  an  organism,  we  still  have  no  explanation  why  pathogenic  organ- 
isms may  exist  at  these  points  without,  consequent  infection.  The 
demonstration  of  certain  preferences  of  localization  for  the  growth 
of  an  organism  is,  however,  in  itself  an  important  point  to  establish 
for  unless  local  conditions  were  such  that  the  invader  could  at  least 
maintain  itself,  subsequent  infection  would  of  course  be  rendered 
difficult. 

Infection  from  the  normal  stomach  where  hydrochloric  acid  is 
being  produced  during  many  hours  of  the  day,  would  a  priori  seem 
to  be  a  difficult  matter.  In  consequence  of  the  active  motility  of 


OBSTACLES  TO  INFECTION  25 

the  organ,  however,  some  of  the  microorganisms  which  have  been 
swallowed  may  readily  escape  destruction,  and  on  entering  the 
intestinal  canal,  with  its  alkaline  reaction  and  numerous  nooks  and 
crevices,  find  suitable  conditions  for  active  growth,  food  material 
being  present  in  abundance.  The  main  danger  to  an  invader  would 
then  evidently  come  from  the  numerous  saprophytic  organisms 
which  have  their  normal  habitat  in  the  very  domain  in  which  the 
newly  introduced  organism  is  a  stranger.  As  such  it  might  readily 
be  destroyed  or  overgrown  by  the  others.  If  introduced  in  suffi- 
ciently large  number,  however,  the  invader  could  unquestionably 
maintain  itself,  for  a  while  at  least,  and  actually  become  a  source  of 
danger,  but  be  destroyed  in  the  end  by  the  normal  inhabitants  of 
the  bowel.  On  the  other  hand,  the  organism  might  adjust  itself  to 
its  new  environment,  lose  its  dangerous  properties  in  a  measure,  and 
continue  to  exist  without  harm  to  the  host.  This  is  probably  true 
of  a  number  of  the  inhabitants  of  the  bowel  which  we  look  upon 
as  normal,  such  as  the  colon  bacillus,  certain  streptococci,  staphylo- 
cocci,  and  others. 

Whether  or  not  microorganisms  would  find  the  mouth  and  nasal 
passages  a  favorable  place  for  growth  under  perfectly  normal  con- 
ditions might  very  well  be  questioned.  The  normal  secretions 
which  find  their  way  into  the  mouth  and  nares  are  undoubtedly 
possessed  of  germicidal  properties,  which  are  feeble  to  be  sure,  but 
nevertheless  existent,  and  it  is  doubtful  whether  microorganisms 
could  maintain  themselves  and  multiply  in  those  parts  which  are 
well  irrigated  by  these  secretions.  In  man,  however,  there  are 
many  nooks  and  corners  where  bacteria  may  lodge  and  escape  the 
action  of  the  salivary  and  nasal  secretion.  The  importance  in  this 
connection  of  carious  teeth,  alveolar  disease,  the  crypts  of  the  tonsils, 
and  pharyngeal  and  postnasal  lymphadenoid  structures,  etc.,  can 
hardly  be  over  estimated.  Such  districts  are  notorious  breeding-places 
of  microorganisms,  and  recognized  portals  of  infection.  But,  after 
all,  while  fully  realizing  that  infection  is  more  apt  to  occur  from  cer- 
tain areas  than  from  others,  the  question  still  remains  unanswered, 
Why  is  it  that  invasion  is  not  invariably  followed  by  infection? 

Obstacles  to  Infection. — The  strongest  general  obstacle  to  infection 
no  doubt  lies  in  the  mechanical  integrity  of  the  epithelial  lining  of 
the  surface  of  the  body  and  its  cavities  and  ducts,  which  are  in 
direct  or  indirect  communication  with  the  exterior.  This  has  long 


26  THE  NATURE  OF  INFECTION 

been  recognized  and  is  well  established.  Organisms  like  the  staphy- 
lococcus  and  the  streptococcus  can  thus  exist  on  the  intact  skin  with- 
out giving  rise  to  any  disturbance  whatever;  they  are  evidently 
unable  to  penetrate  to  the  deeper  structures  through  their  own 
efforts.  The  same  manifestly  holds  good  for  the  epithelial  struc- 
tures of  the  body  in  general;  staphylococci  and  streptococci  exist  in 
the  intestinal  tract  as  on  the  surface  of  the  body  without  causing 
any  damage.  If,  however,  the  epithelial  covering  at  any  point  is 
broken  and  invasion  at  the  point  of  injury  has  preceded,  or  has 
occurred  at  the  same  time,  infection  of  greater  or  less  extent  will  of 
necessity  follow.  In  many  instances  of  infection  with  the  organisms 
in  question  the  break  in  the  continuity  of  the  epithelial  covering 
may  be  ever  so  slight,  but  it  is  very  doubtful  whether  infection  with 
these  organisms  ever  occurs  through  an  intact  epithelial  barrier. 

Various  attempts  have  been  made  to  prove  that  infection  with 
other  organisms  can  take  place  in  this  manner,  but  the  experiments 
do  not  carry  complete  conviction.  It  has  thus  been  argued  that  the 
intact  skin  must  be  permeable  to  an  organism  like  the  plague  bacillus, 
because  the  disease  invariably  develops  in  guinea-pigs  in  which  the 
organism  has  been  rubbed  into  the  shaved  abdominal  surface.  Such 
experiments  demonstrate,  of  course,  that  infection  with  the  organism 
in  question  may  be  effected  through  the  skin ;  but  they  do  not  prove 
by  any  means  that  infection  takes  place  through  the  intact  skin. 
Through  the  mere  process  of  rubbing  slight  injuries  are  unquestion- 
ably produced,  if  not  directly,  then  at  least  indirectly,  for  we  can 
readily  see  that  the  occlusion  of  hair  follicles  and  the  ducts  of  sweat 
glands  by  little  plugs  of  bacteria  constitutes  an  injury  just  as  well  as 
a  visible  abrasion  of  the  surface.  Such  little  plugs  of  bacteria  may, 
on  the  one  hand,  act  as  foreign  bodies  and  mechanically  damage  the 
more  delicate  structures  with  which  they  come  in  contact,  or  the 
bacteria  as  such,  through  their  own  secretory  or  degenerative  prod- 
ucts may  cause  a  local  destruction  of  these  more  delicate  structure 
and  thus  open  a  route  to  infection  of  the  underlying  tissues.  This 
possibility  must  also  be  considered  in  cases  where  infection  takes 
place  apparently  directly  from  the  very  surface  of  epithelial  linings, 
but  is  naturally  less  likely  to  play  a  role  in  so  dense  a  structure  as 
the  surface  epithelium  of  the  skin,  as  in  the  case  of  the  mucous 
membranes. 

The  infection  of  the  urethral  mucosa  by  the  gonococcus  is  fre- 


OBSTACLES  TO  INFECTION  27 

quently  cited  as  an  example  of  the  possibility  of  infection  through 
intact  epithelium.  But  we  know  that  filtrates  of  gonococcus  cultures 
are  in  themselves  capable  of  producing  a  mucopurulent  inflammation 
of  the  human  urethral  mucous  membrane,  so  that  here  again  no 
proof  has  been  afforded  that  the  organism  can  infect  through  intact 
epithelium.  It  is  true  that  the  bacterium  itself  seems  to  be  capable 
of  causing  the  break  in  the  integrity  of  the  epithelium  through  its 
own  products,  but  there  is  also  ground  for  the  belief  that  this  break 
must  first  occur  before  actual  infection  can  take  place.  With  such 
an  organism  as  the  gonococcus  the  question  may  of  course  be  right- 
fully asked  whether  it  can  ever  occur  on  the  mucosa  of  the  urethra 
without  causing  infection;  in  other  words,  whether  primary  invasion 
may  ever  occur  without  being  followed  by  infection.  The  possibility 
unquestionably  exists,  and  we  may  then  conceive  that  the  organism, 
in  order  to  multiply  sufficiently  to  cause  damage  by  its  own  products, 
must  first  find  a  suitable  soil  for  its  growth,  and  that  this  soil  may 
after  all  be  furnished  at  some  break  in  the  continuity  of  the  epithelial 
covering. 

In  the  case  of  the  diphtheria  bacillus  this  is  very  likely,  for  we  know 
that  the  toxin  production  of  this  organism  is  fairly  constant  and  that 
there  is  no  ground  for  believing  that  toxin  is  not  formed  by  those 
bacilli  which  may  at  times  be  found  in  the  throats  of  perfectly 
healthy  individuals.  As  the  amount  of  toxin  is,  however,  evidently 
not  sufficient  to  cause  local  necrosis,  we  must  assume  that  conditions 
for  active  multiplication  of  the  organism  are  not  favorable,  and  we 
can  readily  believe  that  a  break  in  the  continuity  of  the  epithelium 
at  some  point  might  be  the  essential  factor  which  would  lead  to  an 
actual  infection.  This  break  may  occur  through  mechanical  means, 
but  it  may  also  occur  through  the  intervention  of  associated  patho- 
genic agents,  and  I  would  emphasize  particularly  the  importance 
of  underlying  pyogenic  infections,  for  the  occurrence  of  which  the 
ground  is  especially  favorable  in  the  lymphadenoid  structures  of  the 
fauces  and  the  nasopharynx. 

The  importance  of  such  associated  infections  cannot  be  over- 
estimated. We  have  good  evidence  to  show  that  in  their  absence, 
infection  with  certain  bacteria  cannot  occur  at  all.  It  is  thus  well 
known  that  the  tetanus  bacillus  cannot  maintain  itself,  when  in- 
oculated by  itself  into  perfectly  normal  tissues,  while  the  simultaneous 
introduction  of  pyogenic  organisms  renders  its  growth  and  multi- 


28  THE  NATURE  OF  INFECTION 

plication  possible.  In  the  case  of  the  diphtheria  bacillus  similar  con- 
siderations may  apply.  It  must  be  admitted,  however,  that  mechanical 
injury  is  of  paramount  importance,  for  we  see  that  the  tetanus 
bacillus,  while  unable  to  grow  and  multiply  in  structures  that  are 
intact,  can  do  so  when  these  have  been  previously  or  simultaneously 
bruised  or  lacerated.  For  the  reason  that  the  two  organisms  in 
question  can  only  exist  to  advantage  in  damaged  structures,  Bail 
not  inappropriately  speaks  of  them  as  necroparasites  (necros — dead) . 


CHAPTER  III 

THE  OFFENSIVE  FORCES  OF  THE  INVADING 
MICROORGANISM 

WHILE  the  protection  which  the  macro  organism  is  afforded  by  its 
epithelial  covering  is  thus  undoubtedly  of  great  importance,  it  is  a 
striking  fact  that  infections  of  serious  extent  are  after  all  compara- 
tively rare,  if  we  consider  the  frequency  with  which  injury  of  the 
tegumentary  and  mucous  surfaces  occur.  Minor  infections,  on  the 
other  hand,  are  common  enough,  and  the  question  naturally  arises, 
Why  does  not  every  infection  become  generalized  and  lead  to  the 
destruction  of  the  host?  Evidently  this  must  depend  upon  one  of 
two  factors  (sc.,  an  interaction  between  the  two),  viz.,  the  nature 
of  the  microorganism  and  the  resistance  which  the  macro  organism 
offers  to  the  presence  of  the  other.  Collectively  those  forces  which 
are  at  the  disposal  of  the  invading  organism,  and  in  virtue  of  which 
it  strives  to  maintain  itself  in  its  new  environment,  may  be  termed 
its  aggressive  forces,  in  contradistinction  to  the  defensive  forces  of 
the  host.  The  former  will  occupy  our  attention  in  the  present 
chapter. 

Necroparasites. — Bacteriological  examination  of  the  blood  during 
the  life  of  the  patient,  and  of  the  various  tissues  after  death,  reveals 
a  remarkable  difference  in  infections  with  different  organisms.  We 
thus  find  that  certain  bacteria,  such  as  the  diphtheria  bacillus,  the 
tetanus  bacillus,  and  the  bacillus  botulinus,  are  possessed  of  a  very 
low  grade  of  infectiousness,  if  by  this  term  we  mean  their  power 
to  multiply  in  the  invaded  organism.  The  infection  is  almost 
always  strictly  local  during  the  life  of  the  patient;  a  general  infection 
is  indeed  exceedingly  rare,  and  when  it  occurs  it  does  so  only  sub 
finem  vita  or  after  the  death  of  the  patient.  The  tetanus  bacillus 
particularly  is  practically  unable  to  maintain  itself  in  normal  living 
tissues,  and  in  cases  of  infection  owes  its  limited  development  either 
to  the  damage  done  by  an  associated  infecting  agent  or  by  direct 
mechanical  injury.  Even  so,  the  organism  has  frequently  disappeared 


30  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

from  the  body  entirely,  at  the  time  when  the  patient  is  actually 
dying  from  the  effects  of  its  brief  sojourn.  Evidently  its  aggressive 
forces  are  minimal,  and  even  though  it  kills  through  its  highly 
poisonous  toxin,  the  resistance  which  the  animal  body  offers  to  its 
presence  is  entirely  sufficient  to  prevent  its  active  development. 

In  the  case  of  the  diphtheria  bacillus  similar  considerations  apply, 
although  the  organism,  after  once  it  has  gained  a  foothold,  is  not 
dependent  to  the  same  extent  upon  outside  factors  for  its  existence 
in  the  tissues.  It  may  be  questionable  whether  it  can  gain  access  to 
the  deeper  tissues  through  intact  superficial  structures,  but  through 
its  own  toxin  it  is  evidently  capable  of  causing  marked  destruction 
after  once  the  superficial  epithelial  barrier  has  been  passed.  Asso- 
ciated pyogenic  organisms  undoubtedly  facilitate  its  growth,  but  in 
the  deeper  structures,  at  least,  their  cooperation  is  not  imperative. 
The  diphtheritic  exudate  may  of  course  extend  considerably  beyond 
the  original  focus  of  infection,  but  the  infection  after  all  remains 
a  local  one  in  the  vast  majority  of  cases.  If  it  becomes  generalized 
at  all,  this  occurs  well  along  toward  the  fatal  end  or  after  death, 
and  is  even  then  usually  insignificant.  In  the  case  of  this  organism 
also  the  aggressivity  is  thus  not,  as  a  rule,  capable  of  overcoming 
the  defensive  forces  of  the  body,  while  at  the  same  time  it  is  highly 
dangerous  through  its  toxin.  Evidently  the  infectious  and  toxic 
properties  of  an  organism  are  two  independent  factors  which  in  the 
case  of  the  tetanus  and  diphtheria  bacilli  bear  an  inverse  relation 
to  each  other. 

True  Parasites. — An  altogether  different  behavior  is  seen  in  a  group 
of  organisms  which  is  represented  by  the  anthrax  bacillus  and  the 
chicken  cholera  bacillus.  Here  the  local  infection  is  followed  almost 
immediately  by  a  generalized  infection,  the  organisms  not  only 
maintaining  themselves,  but  actually  multiplying  freely  in  the  body 
of  the  host.  Their  aggressivity,  as  compared  with  the  so-called 
necroparasites,  is  thus  extraordinarily  developed,  while  their  toxicity 
is  virtually  nil.  Manifestations  of  disease  are  notoriously  lacking, 
while  death  nevertheless  follows.  It  is  remarkable  to  see  rabbits  or 
sheep  infected  with  anthrax  bacilli,  whose  blood  is  literally  swarm- 
ing with  these  organisms,  quietly  feeding  and  then  dying  a  sudden 
death  without  any  previous  manifestations  of  disease.  We  may 
similarly  see  guinea-pigs  which  have  been  partially  immunised 
against  chicken  cholera,  with  the  peritoneal  cavity  a  veritable  culture 


TRANSITION  FORMS  31 

of  the  organism,  without  any  evidence  of  disease.  Such  examples 
form  the  exact  counterpart  to  what  we  see  in  the  case  of  the 
necroparasites,  but  here  as  there  toxicity  and  infectiousness  or 
aggressivity  bear  an  inverse  relation  to  each  other.  We  see,  more- 
over, that  clinical  manifestations  of  disease  may  be  most  pronounced 
on  the  one  hand,  even  though  the  infecting  organisms  have  been 
unable  to  maintain  themselves  in  the  body  of  the  host  (tetanus), 
while,  on  the  other,  there  may  be  the  most  extensive  infection  with- 
out any  evidence  of  a  corresponding  infectious  disease.  As  Bail 
has  suggested,  organisms  belonging  to  this  latter  class  may  well 
be  looked  upon  as  true  parasites,  whose  aggressive  mechanism  must 
evidently  be  of  a  different  nature  than  that  of  the  necroparasites 
previously  considered. 

Semiparasites. — Between  these  two  extremes  stand  the  semi- 
parasites,  which  are  represented  by  the  cholera  vibrio  and  the  typhoid 
bacillus.  Their  infectiousness,  and  hence  aggressivity,  is  already 
quite  well  developed,  although  it  is  not  comparable  to  what  we  see 
in  anthrax  or  chicken  cholera,  necessitating  (in  the  animal  experi- 
ment) the  introduction  of  a  fairly  large  number  of  organisms  and 
often  special  methods  of  infection.  In  man  the  typhoid  bacillus  is 
distinctly  more  aggressive  than  the  cholera  vibrio,  which  latter  is 
rarely  found  in  the  blood  or  tissues,  although  one  would  imagine, 
in  view  of  the  extensive  epithelial  desquamation  and  superficial 
necroses,  that  opportunity  for  a  general  invasion  would  be  readily 
afforded.  In  addition  to  their  aggressiveness  the  organisms  of  this 
class  are  possessed  of  a  well-marked  toxicity,  the  effect  of  which 
appears  quite  early  in  the  course  of  the  infection,  but  does  not  lead 
to  the  production  of  specific  symptoms,  as  we  see  them  in  the  case 
of  the  necroparasites. 

Transition  Forms. — From  the  semiparasites  the  transition  to  the 
necroparasites  is  represented  by  certain  anaerobic  butyric  acid 
producing  bacilli,  such  as  the  bacillus  of  malignant  edema  and  the 
bacillus  of  symptomatic  anthrax,  which  are  actively  necrotizing 
toxin  producers,  but  possess  a  certain  degree  of  aggressivity  also, 
as  is  evidenced  by  their  wider  distribution  in  the  body  of  the  infected 
animal.  Next  in  order  follows  the  dysentery  bacillus  which  behaves 
as  a  typical  semiparasite  for  the  guinea-pig,  while  in  the  rabbit  it  is 
relatively  little  infectious  but  exhibits  a  marked  toxin  production. 
The  streptococcus  and  pneumococcus,  on  the  other  hand,  are  closely 


32  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

related  to  the  true  parasites,  being  characterized  by  a  considerable 
degree  of  infectiousness  and  a  low  grade  of  toxicity. 

The  remaining  pathogenic  organisms  can  be  readily  placed  in  this 
system,  the  determining  factors  being  their  aggressivity  (infectious- 
ness)  and  their  toxicity.  The  plague  bacillus  would  thus  find  its 
proper  position  close  to  the  true  parasites,  while  the  staphylococcus, 
meningococcus,  and  gonococcus  would  come  somewhere  between 
the  staphylococcus-pneumococcus  group  and  the  semiparasites 
proper,  and  so  on.  It  should  be  borne  in  mind,  however,  that  the 
exact  position  of  an  organism  in  this  system  may  vary  with  different 
species  of  animals,  at  least  so  far  as  its  aggressivity  is  concerned. 
I  have  pointed  out  already  that  the  position  given  the  cholera 
bacillus,  for  example,  is  not  exactly  correct  in  the  case  of  man, 
where  it  should  stand  close  to  the  necroparasites.  The  anthrax 
bacillus  in  the  frog  and  pigeon  has  ordinarily  no  aggressivity  what- 
ever, even  though  the  same  strain  may  be  most  active  in  other  mam- 
mals. The  factors  which  produce  this  difference  in  behavior  are 
frequently  unknown,  but  sometimes,  as  in  the  last  example,  they 
are  very  simple;  for  in  this  instance  the  apparently  absolute 
resistance  of  the  frog  and  pigeon  is  referable  to  the  fact  that  the 
anthrax  bacillus  ordinarily  does  not  grow  at  the  temperature  which 
is  normal  for  the  animals  in  question.  If,  however,  one  gradually 
accustoms  the  organism  to  those  temperatures  infection  can  be 
produced. 

Tissue  Parasites. — It  will  be  noted  that  no  mention  has  been  made 
of  the  position  of  either  the  tubercle  bacillus  or  of  actinomyces  in 
the  above  schema.  As  a  matter  of  fact,  these  organisms  occupy 
a  position  of  their  own,  being  essentially  tissue  parasites,  while  the 
others  may  be  looked  upon  as  humoral  parasites.  Their  behavior 
in  the  macro  organism  is  in  every  respect  different  from  that  of  the 
remainder.  While  the  latter  only  affect  a  certain  group  of  cells 
(i.  e.,  the  leukocytes)  in  a  direct  manner,  the  tissue  parasites  bring 
about  an  altered  response  of  the  body  at  large,  i.  e.,  an  allergia 
which  is  in  a  certain  sense  characteristic  of  this  group.  This  peculiar 
behavior  is  also  shown  by  one  of  the  animal  parasites,  viz.,  the 
treponema  pallidum,  while  the  trypanosomes  resemble  the  humoral 
bacterial  parasites,  (see  also  section  on  Allergia). 

Virulence,  Infectiousness,  and  Aggressivity. — From  the  foregoing 
survey  it  is  clear  that  the  aggressivity  of  the  pathogenic  organisms 


VIRULENCE,  INFECTIOUSNESS,  AND  AGGRESSIVITY       33 

differs  very  considerably,  and  the  question  naturally  arises,  To  what 
is  this  difference  due? 

Clinically,  we  have  long  been  in  the  habit  of  ascribing  the  varying 
severity  observed  in  different  cases  of  the  various  infectious  diseases 
to  differences  in  the  virulence  of  the  organism;  in  other  words,  the 
severity  of  the  clinical  picture  was  regarded  as  an  index  of  the  severity 
of  the  infection.  This  conception  of  the  term  is  no  longer  tenable, 
in  view  of  our  present  knowledge  of  the  relation  or  rather  lack  of 
relation  which  exists  between  infection  and  infectious  diseases;  for, 
as  we  have  seen,  the  anthrax  bacillus  produces  no  evidence  of  disease 
whatever  until  the  end  is  almost  at  hand,  although  the  blood  may 
be  swarming  with  organisms  long  before. 

Using  the  term  virulence  in  the  old  sense  of  the  word,  we  would 
accordingly  be  forced  to  look  upon  every  anthrax  infection  as  a  non- 
virulent  infection,  which  would  evidently  be  absurd.  On  the  other 
hand,  we  know  that  in  tetanus  serious  symptoms  of  disease  appear 
relatively  early,  even  though  the  bacillus  multiplies  to  a  slight  extent 
only,  and  the  infection  remains  altogether  local;  in  some  cases, 
indeed,  the  organisms  have  already  disappeared  from  the  body  at  a 
time  when  the  patient  is  dying  from  the  effect  of  their  toxins.  Such 
an  infection  one  would  be  apt  to  look  upon  as  especially  virulent. 
Evidently  the  severity  of  the  clinical  pictures  is  no  index  of  the  vir- 
ulence of  the  organism.  The  confusion  is  altogether  due  to  the  fact 
that  in  the  past  the  toxic  power  of  an  organism  and  its  infectious 
power  have  been  looked  upon  as  synonymous,  while  we  now  recog- 
nize that  the  two  are  separate  factors. 

The  toxicity  of  an  organism  is  in  a  measure  an  accidental  property 
which  is  of  interest  from  the  fact  that  it  is  responsible  for  certain 
symptoms  of  the  infection,  but  it  is  not  by  any  means  essential  to 
infection.  This  is  shown  especially  well  in  the  case  of  the  tetanus 
bacillus,  wrhose  toxins  by  themselves,  after  separation  from  the 
organism,  are  capable  of  producing  the  identical  clinical  picture 
which  follows  actual  infection. 

The  term  virulence,  in  its  modern  meaning,  has  reference  essen- 
tially to  the  ability  of  an  organism  to  multiply  in  the  body  of  the 
infected  animal,  and  is  hence  virtually  synonymous  with  infectiousness 
or  aggressivity.  It  is,  hence,  erroneous  to  speak  of  the  virulence  of  a 
tetanus  bacillus  or  a  diphtheria  bacillus  to  indicate  the  severity 
of  a  given  case;  the  clinical  picture  is  essentially  due  to  the  action 
3 


34  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

of  toxins,  and  one  should  accordingly  speak  of  the  toxicity  of  the 
organism.  Its  virulence,  i.  e.,  its  power  to  multiply  in  the  body  of  the 
infected  animal,  is  always  slight.  Death  is  the  outcome  of  its  toxic 
action,  but  not  the  expression  of  an  especially  high  degree  of  virulence. 

The  use  of  the  latter  term  in  infections  with  the  necroparasites 
would  only  be  justifiable  if  one  wished  to  give  expression  to  the 
idea  that  the  severity  of  the  clinical  picture  was  due  to  the  forma- 
tion of  an  especially  large  quantity  of  toxin,  which  in  turn  would 
indicate  the  presence  of  an  especially  large  number  of  organisms. 
This,  however,  scarcely  enters  into  consideration,  as  we  know  that 
the  necroparasitic  toxins  are  so  extremely  active  that  large  numbers 
of  organisms  are  not  at  all  needed  to  produce  disastrous  consequences, 
after  primary  infection  has  once  taken  place. 

In  infections  with  the  true  parasites,  on  the  other  hand,  the  term 
virulence  in  its  new  sense  is  directly  applicable;  the  more  virulent 
the  organism  the  more  readily  will  it  multiply  in  the  body  of  the 
infected  animal.  In  the  semiparasites  the  term  virulence  may 
occasionally  still  be  applied  in  its  original  meaning,  and  the  more 
justifiably  so  the  more  evenly  the  toxic  and  the  infectious  properties 
are  represented  in  the  same  organism.  By  a  particularly  virulent 
infection  we  would  here  mean  an  infection,  during  which  there  is, 
on  the  one  hand,  an  active  multiplication,  and  on  the  other  a 
correspondingly  active  toxin  formation  with  the  production  of 
a  correspondingly  severe  clinical  picture. 

Differences  in  Aggressivity. — Having  thus  established  the  proper 
meaning  of  the  term  virulence  we  may  now  return  to  the  question, 
To  what  factor  is  the  difference  in  the  aggressivity  and  hence  the 
virulence  of  the  different  groups  or  strains  of  bacteria  due?  Two 
possibilities  naturally  suggest  themselves,  which  may  be  operative 
either  individually  or  conjointly.  On  the  one  hand  we  may  imagine 
that  an  organism  when  introduced  into  the  body  of  an  animal 
which  seeks  to  destroy  the  invader  adjusts  itself  to  its  new  sur- 
roundings by  certain  changes  of  a  morphological  or  physiological 
character,  in  consequence  of  which  it  becomes  relatively  or  absolutely 
unassailable  by  the  offensive  forces  of  the  host,  unless,  indeed,  it 
already  possesses  such  properties  during  its  saprophytic  existence 
outside  of  the  body.  On  the  other  hand  we  can  conceive  that  the 
infecting  organism  actively  secretes  material  which  tends  to  counter- 
act or  even  to  destroy  the  opposing  forces  of  the  host. 


PASSIVE  AGGRESSIVITY  35 

Aggressins. — Such  substances  Bail  has  termed  aggressins,  and  he 
speaks  of  aggressivity  of  this  order  as  aggressivity  in  the  narrower 
sense,  while  he  denotes  the  former  as  aggressivity  in  the  wider  sense  of 
the  term.  In  their  places  one  could  substitute  the  terms  active  and 
passive  aggressivity,  the  latter  indicating  a  passive  resistance  and  the 
former  an  actual  offensive  reaction.  The  general  recognition  of  the 
existence  of  a  certain  aggressivity  on  the  part  of  the  invading  organ- 
ism is  most  important.  If  in  the  past  the  attention  of  medical  men 
has  been  centred  on  the  defensive  mechanism  of  the  invaded  organism 
this  interest  has  been  essentially  a  selfish  one.  Active  progress  in 
the  future,  however,  will  depend  to  a  considerable  degree  upon  our 
knowledge  of  the  defensive  forces  of  the  invader.  Our  present 
knowledge  is  as  yet  quite  small,  but  enough  has  been  learned  to 
establish  the  importance  of  further  research  in  this  direction. 

Passive  Aggressivity. — CAPSULE  FORMATION. — Among  the  passive 
factors  the  most  striking  is  the  tendency  to  capsule  formation,  which 
occurs  in  some  of  the  pathogenic  bacteria,  while  they  exist  in  the 
animal  body,  or  when  they  are  grown  on  media  containing  animal 
albumins,  such  as  serum,  serum  agar,  hydrocele  agar,  milk,  etc. 
When  the  organism  is  transplanted  to  ordinary  media  this  peculiarity 
rapidly  disappears,  but  can  be  made  to  reappear  by  transferring  it 
to  albuminous  media,  or  by  reinoculation  into  the  animal  body, 
and  so  on  indefinitely.  The  most  notable  organisms  which  possess 
this  property,  aside  from  the  capsule  bacteria  proper,  viz.,  those 
organisms  which  even  under  ordinary  circumstances  possess  a  cap- 
sule, such  as  the  bacillus  pneumonise  of  Friedlander,  and  the  bacillus 
of  rhinoscleroma,  are  a  number  of  streptococci  (str.  involutus, 
vulvitidis  vaccarum,  mastitidis  vaccarum,  equi,  mucosus),  the  pneu- 
mococcus,  the  micrococcus  tetragenus,  bacterium  anthracis,  bacterium 
pestis,  bacterium  cholerse  gallinarum,  certain  pathogenic  yeasts,  etc. 
Coincidently  there  seems  to  be  an  absence  of  that  tendency  to  form 
chains  which  is  so  common  in  the  case  of  certain  organisms,  such  as 
the  anthrax  bacillus  and  certain  streptococci  and  pneumococci, 
when  these  are  grown  on  artificial  media. 

In  other  organisms  actual  capsule  formation  has  not  been  observed 
but  in  its  place  an  analogous  process  has  been  noted,  resulting  in 
a  thickening  of  the  ectoplasm,  so  that  the  bacteria  look  larger  and 
coarser.  This  is  true  especially  of  the  colon  and  typhoid  bacillus 
and  of  the  staphylococcus,  and  leads  to  appearances  which  often 


36  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

contrast  strongly  with  the  tiny  attenuated  forms  which  one  is  accus- 
tomed to  see  in  old  cultures  on  the  ordinary  media. 

The  importance  of  these  morphological  changes  as  a  defensive 
mechanism  of  the  bacteria  against  the  opposing  forces  of  the  host 
can  hardly  be  overestimated.  It  has  been  conclusively  demon- 
strated, as  a  matter  of  fact,  that  such  "  animalized"  bacteria,  as 
Bail  terms  them,  offer  a  far  greater  resistance  to  the  destructive 
action  of  bactericidal  sera  and  to  phagocytosis  than  do  the  corre- 
sponding forms  which  have  been  cultivated  on  the  ordinary  media. 
That  such  changes  must  of  necessity  lead  to  a  marked  increase 
in  the  virulence  of  an  organism  is  of  course  self-evident.  This 
is  well  illustrated  by  an  experiment  of  Horiuchi,  who  relates  that 
he  had  in  his  possession  a  highly  virulent,  densely  capsulated  strain 
of  the  micrococcus  tetragenus,  which  resisted  phagocytosis  almost 
entirely  and  killed  guinea-pigs  in  a  dose  of  100  organisms.  When 
this  was  grown  for  a  number  of  days  on  rather  dry  agar  it  lost  its 
capsule-forming  power  permanently,  became  readily  subject  to 
phagocytosis,  and  did  not  affect  guinea-pigs  even  in  doses  of 
1,000,000,000  organisms. 

In  view  of  our  present  knowledge  of  the  relation  between  capsule 
formation  and  virulence,  we  can  now  readily  understand  why  animal 
passage  of  an  organism  leads  to  increased  virulence.  This  fact  had 
long  been  recognized  by  bacteriologists,  but  an  adequate  explana- 
tion for  it  had  long  been  wanting.  By  starting  with  a  laboratory 
culture  that  has  been  grown  for  many  generations  on  artificial, 
non-albuminous  media,  it  may  be  absolutely  impossible  to  produce 
an  infection  at  all,  even  though  enormous  numbers  of  bacteria  be 
injected.  If  injection,  however,  results  we  may  imagine — in  the 
case  of  one  of  the  organisms  in  which  capsule  formation  occurs — 
that  even  though  the  majority  of  organisms  had  lost  the  power 
of  forming  capsules,  and  of  thus  resisting  the  offensive  forces  of  the 
host,  a  certain  number  still  possessed  this  property,  and  that  these 
escaped  destruction  and  multiplied  to  a  greater  or  less  extent. 

If  then  at  the  height  of  the  infection  the  animal  is  killed,  or  if  it 
succumbs  to  the  infection  directly,  the  now  capsulated  bacteria  will 
be  found  capable  of  successfully  infecting  the  next  animal,  to  which 
they  should  be  transferred  without  being  first  replanted  on  ordinary 
media.  As  a  result  of  the  increased  degree  of  resistance  which  the 
organisms  have  acquired  in  the  first  animal,  they  are  now  in  a  much 


PASSIVE  AGGRESSIVITY  37 

better  position  to  maintain  themselves  and  to  multiply  in  the  second, 
and  as  the  transfers  are  continued  through  a  series  of  animals,  it 
will  be  observed  that  the  number  of  organisms  which  is  necessary 
to  kill  the  animal  becomes  progressively  smaller,  and  the  period  of 
incubation,  i.  e.,  the  interval  elapsing  between  infection  and  the  first 
evidence  of  the  resulting  disease,  shorter,  until  finally  a  strain  is 
obtained  in  which  the  degree  of  virulence  can  no  longer  be  increased 
by  animal  passage — this  constitutes  the  virus  fixe  in  the  sense  of 
Pasteur. 

Potential  Virulence. — While  we  have  thus  gained  a  material  basis 
for  our  concept  of  the  actual  virulence  of  an  organism,  it  is  important 
also  to  recognize  a  certain  potential  virulence,  viz.,  the  ability  of  an 
organism  actually  to  form  capsules  when  placed  under  conditions 
which,  cceteris  paribus,  are  favorable  to  their  development.  Evi- 
dently only  those  organisms  of  the  capsule-forming  group,  or  at  any 
rate  those  in  which  an  hypertrophy  of  the  ectoplasm  can  occur,  are 
capable  of  acquiring  a  notable  degree  of  virulence  in  which  this 
potentiality  is  inherent.  If  once  this  is  permanently  lost  the  organism 
in  question  is  manifestly  non-virulent,  so  far  as  its  actual  develop- 
ment in  the  infected  animal  is  concerned.  We  have  had  an  excellent 
illustration  of  this  in  Horiuchi's  experiment,  referred  to  above.  To 
determine  this  potentiality  it  is  sometimes  only  necessary  to  grow 
the  organism  in  serum-containing  media  and  to  examine  micro- 
scopically for  capsules.  In  the  non-capsule  formers,  on  the  other 
hand,  microscopic  examination  is  insufficient  to  determine  whether 
an  organism  under  consideration  is  virulent  or  not;  in  that  case  the 
animal  experiment  alone  will  decide  the  question. 

Factors  Determining  Capsule  Formation  and  its  Significance. — 
Of  the  factors  which  are  operative  in  determining  the  formation  of 
capsules  very  little  is  as  yet  known.  One  could  imagine,  of  course, 
that  as  the  result  of  favorable  changes  in  nutrition,  certain  biological 
changes  would  result  of  which  the  hypertrophy  of  the  ectoplasm 
is  one  of  the  consequences;  in  other  words,  that  capsule  formation 
is  an  index  of  a  condition  of  particularly  active  nutrition.  There 
are  certain  facts,  however,  which  suggest  that  this  explanation  is 
not  correct.  We  find  that  capsule  formation  may  be  evoked  by  agents 
which  have  no  nutrient  properties  whatever.  Danysz  thus  found  that 
the  anthrax  bacillus  when  grown  in  arsenical  media  of  increasing 
concentration  forms  enormous  mucinous  capsules  which  protect 


38     OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

the  organism  against  the  bactericidal  action  of  the  chemical  in 
question. 

Weil  and  Suzuki  further  found  that  certain  sarcinae  (i.  e.,  absolute 
saprophytes)  when  exposed  to  the  action  of  leukocytes  which  readily 
destroy  them,  undergo  a  peculiar  mucinous  degeneration  which 
in  the  beginning  is  directly  comparable,  if  not  identical  with  capsule 
formation,  but  proceeds  beyond  this,  and  ends  with  the  death  of 
the  organism.  In  this  case  the  capsule  formation  is  evidence  of  a 
serious  impairment  of  the  vitality  of  the  cell  and  closely  analogous 
to  the  granular  degeneration  of  vibrios  under  the  influences  of  the 
bactericidal  substances  of  the  serum.  It  does  not  necessarily  follow 
of  course  that  capsule  formation  among  the  pathogenic  parasitic 
organisms  should  likewise  be  a  degeneration  phenomenon,  but  the 
above  observations  suggest  this  possibility,  and  it  will  be  well  in 
future  investigations  to  take  it  into  account. 

It  is  also  quite  in  accord  with  the  present  tendency  to  regard 
capsule  formation  as  a  pathological  state  on  the  part  of  the  micro- 
organisms that  the  anthrax  bacillus  never  forms  spores  in  the  animal 
body,  no  matter  how  extreme  the  infection  may  be.  One  would 
accordingly  expect  that  in  a  long-continued  series  of  transplantations 
from  animal  to  animal  the  vitality  of  the  microorganism  would 
finally  be  damaged  so  severely  that  a  further  transfer  would  not 
lead  to  infection.  This  actually  seems  to  be  the  case,  for  neither 
Bail  nor  Gruber  and  Wiener  were  able  to  maintain  an  uninterrupted 
series  in  the  case  of  the  anthrax  bacillus  on  the  one  hand  and  the 
cholera  vibrio  on  the  other. 

In  this  connection  it  is  interesting  to  note  that  in  especially  severe 
infections  certain  organisms,  such  as  the  anthrax  and  the  Friedlander 
bacillus,  may  appear  capsule-free  even  in  the  infected  body,  which  at 
first  sight  is  difficult  to  reconcile  with  the  idea  that  capsule  forma- 
tion is  essential  to  infection.  The  true  significance  of  the  phenomenon, 
however,  is  suggested  by  the  observation  that  in  the  test-tube  experi- 
ment a  serum  may  be  deprived  of  its  power  to  elicit  capsule  formation, 
if  an  abundant  culture  has  once  been  raised  from  it,  which  seems  to 
indicate  that  a  certain  component  which  is  essential  to  capsule 
formation  has  thus  been  removed.  It  is  similarly  possible  in  the 
case  of  certain  strains  of  streptococci  to  produce  either  capsulated 
or  non-capsulated  generations  by  varying  the  intensity  of  the 
infection.  If  then  a  non-capsulated  lot  is  transferred  from  the  body 


PASSIVE  AGGRESSIVITY  39 

of  the  infected  animal  to  a  tube  of  serum  typically  capsulated  organ- 
isms will  develop,  while  the  same  strain,  if  grown  outside  of  the  body 
in  non-albuminous  media,  hardly  shows  any  evidence  of  capsule 
formation  on  being  transferred  to  serum.  The  conclusion  hence 
suggests  itself  that  capsule  formation  is  merely  a  coincidental  evi- 
dence of  a  special  state  which  the  organism  assumes  in  the  animal 
body  and  that  its  increased  resistance  in  the  body  is  not  due  solely 
to  its  capsule,  but  to  the  development  of  a  general  infectious  ability, 
of  which  capsule  formation  and  resistance  to  phagocytosis  are  asso- 
ciated, but  not  necessarily  interdependent  consequences.  Similarly, 
attenuated  organisms  of  this  order  are  harmless,  not  merely  because 
they  have  lost  the  power  to  form  capsules,  but  because  they  are  no 
longer  able  to  assume  that  special  infectious  state  which  may  be 
associated  with  capsule  formation.  We  could  accordingly  conceive 
the  existence  of  a  special  type  of  immunity  which  we  may  term 
antiblastic  immunity  (Ascoli),  which  would  depend  upon  such  an 
inability  of  an  organism  to  develop  an  infectious  state,  even  in 
the  absence  of  any  active  antibacterial  agencies  on  the  part  of  the 
macro  organism. 

Organ  Virulence. — After  the  virulence  of  an  organism  has  been 
artificially  raised,  one  would  imagine  that  this  increase  would  mani- 
fest itself  not  only  in  animals  of  the  same  species  through  which  it 
has  been  passed,  but  in  others  as  well.  This,  however,  is  not  the 
case,  and  here  as  elsewhere  in  immunological  work  one  meets  with 
remarkable  examples  of  specificity  for  which  no  explanation  can  as 
yet  be  given.  If,  for  example,  the  virulence  of  the  chicken  cholera 
bacillus  is  increased  by  passage  through  the  chicken,  this  increase 
affects  this  animal  but  remains  unchanged  for  the  guinea-pig.  Simi- 
larly a  certain  selective  affinity  develops  for  certain  organs  if  the 
increase  in  virulence  has  been  brought  about  through  the  specific 
intervention  of  those  organs,  and  then  shows  itself  irrespective  of  the 
manner  in  which  infection  is  produced.  When  rats,  for  example, 
have  been  serially  infected  through  the  respiratory  tract  with  the 
lung  juice  of  animals  dead  with  the  plague,  the  virulence  of  the 
organisms  is  not  only  increased,  but  plague  pneumonia  invariably 
develops  on  infecting  other  rats  even  by  the  subcutaneous  or  intra- 
peritoneal  route. 

Virulence  of  this  order  which  is  specifically  directed  against  cer- 
tain organs  is  spoken  of  as  organ  virulence  and  is  evidently  destined 


40  OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

to  play  an  important  role  in  the  future  study  of  infection.  Capsule 
formation,  however,  can  scarcely  have  anything  to  do  with  this 
phenomenon  in  itself,  while  we  can  well  imagine  that  nutritional 
factors  may  play  an  important  role.  We  can  thus  conceive  that 
during  the  primary  infection,  which  in  turn  may  be  possible  owing  to 
capsule  formation,  a  certain  group  of  organisms  may  have  become 
lodged  in  a  certain  organ,  and  that  their  vegetative  functions  here 
become  so  modified  that  the  particular  juices  which  are  there  avail- 
able can  be  utilized  especially  well.  If,  then,  members  of  this  strain 
are  subsequently  introduced  into  another  animal,  those  will  develop 
with  special  readiness  which  are  placed  in  contact  with  the  same 
nutriment  to  which  they  had  become  accustomed  in  the  first  host, 
while  the  remainder,  from  lack  of  this  special  nutriment,  may  not 
develop  at  all.  As  a  consequence  that  organ  will  become  the  special 
seat  of  infection  and  disease  in  which  conditions  for  the  growth  of 
the  organism  are  most  favorable.  The  affinity  for  such  an  organ, 
may  of  course  be  a  natural  one,  and  exist  already  on  the  part  of  an 
organism  which  has  not  been  passed  through  an  animal  for  many 
generations,  but  there  can  be  no  doubt  that  it  may  also  be  acquired. 
Attenuation. — The  influence  of  animal  passage  upon  the  aggres- 
sivity  of  an  organism  can  thus  be  twofold — i.  e.,  it  may  lead  to  cap- 
sule formation,  on  the  one  hand,  and  to  a  general  increase  in  its 
functional  efficacy  as  a  consequence  of  especially  favorable  nutri- 
tional conditions,  on  the  other,  the  outcome  being  an  increased 
virulence  for  the  infected  animal.  The  reverse  will  be  caused  by 
those  agencies  which  prevent  the  development  of  these  aggressive 
forces.  We  have  already  pointed  out  that  the  ability  to  form 
capsules  disappears  when  an  organism  is  grown  on  ordinary  media, 
and  we  know  that  this  inability  may  become  permanent;  this  in 
itself  does  not  interfere  with  the  viability  of  the  organism  as  a 
saprophyte,  to  be  sure,  but  makes  its  parasitic  existence  in  the 
animal  body  an  impossibility.  Such  a  decrease  in  the  virulence  of 
an  organism  can  be  brought  about  in  many  other  ways,  although  it 
has  not  been  ascertained  to  what  extent  impaired  capsule  formation 
is  responsible  for  the  change;  in  some  instances  this  may  be  the 
case,  while  in  others  this  explanation  is  hardly  admissible.  Such 
attenuation  in  virulence  can  be  brought  about  by  exposure  to  tem- 
peratures which  are  unfavorable  to  the  growth  of  the  organism; 
prolonged  exposure  to  the  air;  exposure  to  sunlight;  increased  atmos- 


ACTIVE  AGGRESSIVITY  41 

pheric  pressure;  an  electric  current;  certain  chemicals,  such  as 
glycerin,  carbolic  acid,  chlorin,  trichloride  of  iodin,  potassium  bichro- 
mate, alcohol,  etc.,  special  care  being  taken,  of  course,  to  employ 
concentrations  which  will  not  actually  kill  the  organisms;  further, 
by  growing  an  organism  in  the  presence  of  others  which  tend  to 
crowd  out  the  one  under  consideration;  by  growth  in  immune 
serum,  etc. 

One  additional  method  deserves  consideration,  as  on  first  thought 
its  employment  might  be  expected  to  lead  to  an  increase  in  virulence 
instead  of  the  reverse — namely,  animal  passage.  We  have  pointed  out 
before  that  the  virulence  of  an  organism  is  thus  usually  specifically 
increased  for  the  species  employed,  while  it  remains  unchanged 
for  other  animals;  it  may  happen,  however,  that  this  one-sided 
increase  is  associated  with  an  actual  decrease  in  virulence  for  other 
species.  We  have  a  practical  application  of  this  principle  in  the 
attenuation  of  the  variola  virus  by  passage  through  the  heifer 
(Jenner),  and  in  Pasteur's  immunization  against  hog  cholera  by 
passing  the  organism  through  rabbits  (weaker  vaccine  I)  and  pigeons 
(stronger  vaccine  II) . 

Most  important  from  a  practical  standpoint  is  the  fact  that 
organisms  which  have  been  attenuated  in  their  virulence  through 
one  of  the  methods  enumerated,  or  through  still  others,  that  have 
for  their  primary  object  a  direct  impairment  of  the  organism's 
resistance,  will  either  not  be  able  to  bring  about  an  infection  at  all,  or 
if  this  does  occur,  a  modified  infection  is  the  outcome  with  the  estab- 
lishment of  a  temporary  or  permanent  immunity — a  phase  of  our 
problem  which  will  be  dealt  with  in  greater  detail  in  a  later  chapter. 
The  essential  point  to  be  borne  in  mind  at  present  is  the  fact  that 
just  as  it  is  possible  by  artificial  means  to  increase  the  virulence 
of  an  organism,  and  thus  to  favor  the  development  of  infection,  so 
also  is  it  possible  to  bring  about  the  reverse,  and  that  the  occurrence 
or  non-occurrence  of  infection  must  of  necessity  depend  to  a  very 
considerable  extent  upon  the  presence  or  absence  of  certain  aggressive 
forces  on  the  part  of  the  organism,  among  which  the  morphological 
evidence  of  aggressivity  is  especially  striking. 

Active  Aggressivity. — I  have  pointed  out  previously  that  in  addi- 
tion to  such  passively  aggressive  forces  it  is  quite  conceivable  that 
microorganisms  may  also  possess  certain  active  forces,  and  a  great 
deal  of  work  has  actually  been  done  in  the  attempt  to  establish  their 


42     OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

existence.  The  true  toxins  would  of  course  suggest  themselves  at 
once  as  such  forces,  but  as  we  have  seen  already,  the  very  organisms 
in  which  toxin  production  is  most  striking  are  the  least  infectious, 
and  they  can  therefore  hardly  enter  into  consideration.  We  have 
thus  shown  that  the  tetanus  bacillus,  for  example,  notwithstanding 
its  active  toxin  production,  is  practically  unable  to  maintain  its 
existence  in  the  body  following  primary  infection.  If,  then,  the 
true  toxins  are  eliminated  as  active  aggressive  forces,  viz.,  as  forces 
which  inhibit  the  action  of  the  offensive  forces  of  the  host,  the  ques- 
tion arises,  What  evidence  have  we  that  such  forces  may  actually 
be  operative? 

With  this  problem  the  work  of  Bail  will  always  remain  intimately 
associated.  This  investigator  found  that  the  peritoneal  exudate  of 
animals  which  had  been  killed  by  intraperitoneal  injection  of  mul- 
tiple fatal  doses  of  such  organisms  as  the  typhoid  and  the  cholera 
bacillus,  upon  subsequent  removal  of  the  organisms  and  sterilization 
of  the  fluid  with  chemical  antiseptics,  was  capable  of  transforming 
subfatal  doses  of  the  same  organism  into  fatal  doses;  in  other 
words,  it  had  acquired  properties  which  evidently  favored  infection. 
Bail  supposed  that  definite  substances  which  were  secreted  by  the 
bacteria  in  the  body  of  the  infected  animal,  and  which  he  termed 
aggressins,  were  concerned  in  the  production  of  this  effect.  He 
assumed  that  the  aggressins  were  substances  sui  generis,  largely 
upon  the  basis  that  aggressive  exudates  in  themselves  were  found 
to  be  non-toxic,  and  when  injected  into  animals  by  themselves 
were  capable  of  preventing  subsequent  infection.  This  he  explained 
by  the  assumption  that  specific  reaction  products  (antibodies)— 
antiaggressins — are  formed  in  consequence  of  the  injection  of  the 
aggressins,  which  render  the  latter  inactive  and  thus  prevent  the 
active  invasion  of  the  body  by  the  microorganisms  in  question 
(antiaggressin  immunity). 

The  aggressive  character  of  the  exudates  is  largely  directed  against 
the  phagocytes,  which,  like  Metschnikoff,  Bail  regards  as  the  only 
true  defensive  elements  of  the  invaded  organism.  This  he  demon- 
strated by  injecting  two  aggressin-immune  animals,  A  and  B, 
intraperitoneally  with  equal  doses  of  a  suitable  number  of  organisms, 
A  receiving,  in  addition,  a  certain  amount  of  aggressin.  After  the 
lapse  of  one  or  two  hours  the  peritoneal  fluid  of  B  can  then  be  shown 
to  contain  large  numbers  of  leukocytes,  and  at  the  expiration  of  four 


ACTIVE  AGGRESSIVITY  43 

hours  the  exudate  is  thick,  tenacious,  milky  looking,  and  is  composed 
almost  entirely  of  polynuclear  leukocytes  which  have  taken  up  many 
or  all  of  the  injected  organisms  according  to  the  number  which  were 
originally  introduced.  In  A,  on  the  other  hand,  the  fluid  is  abundant, 
relatively  clear,  poor  in  cells,  but  swarming  with  organisms,  few  if 
any  of  which  have  been  disposed  of  by  phagocytosis.  Bail's  explana- 
tion is  that  in  B,  where  no  aggressins  have  been  injected,  there  was 
nothing  to  prevent  the  immediate  inroads  of  the  leukocytes,  which 
was  facilitated  in  fact  by  the  immune  condition  of  the  animal,  any 
aggressins  that  were  formed  by  the  bacteria  being  bound  by  the 
antiaggressins  already  present.  In  A,  on  the  other  hand,  the  anti- 
aggressins  were  neutralized  by  the  extra  injection  of  aggressins  as 
such,  which,  moreover,  in  the  presence  of  bacteria,  exercised  their 
negatively  chemotactic  influence  upon  the  leukocytes,  so  that 
bacterial  development  could  go  on  undisturbed.1 

This  interpretation  seems  quite  adequate  to  explain  the  function 
of  the  aggressins  in  infections  with  those  organisms,  which  are 
notoriously  subject  to  phagocytosis,  and  in  which  other  destructive 
agencies  on  the  part  of  the  invaded  animals  play  no  role.  As  will 
be  shown  in  detail  in  Chapter  VI,  however,  there  are  infections,  as 
with  the  cholera  vibrio,  for  example,  in  which  phagocytosis  only  plays 
a  subordinate  role,  but  in  which  the  destruction  of  the  organisms  is 
brought  about  through  certain  bactericidal  substances  (bacteriolysins) 
which  are  present  in  the  serum.  In  such  cases  it  is  at  first  sight 
difficult  to  see  how  the  aggressins  can  play  a  role  at  all,  if,  as  Bail 
suggests,  their  influence  is  directed  almost  entirely  against  the 
leukocytes.  He  has  pointed  out,  however,  that  this  is  the  case, 
nevertheless,  for  it  can  be  shown  that  the  leukocytes  are  capable  of 
rendering  harmless  the  so-called  endotoxins  which  are  liberated  during 
the  solution  of  the  bacteria  (in  consequence  of  the  bactericidal  sc. 
bacteriolytic  property  of  the  serum),  and  that  by  preventing  the 
access  of  the  leukocytes  through  the  agency  of  the  aggressins  the 
animal  succumbs  to  a  final  intoxication. 

As  is  evident  from  the  above-mentioned  facts,  the  possibility  of 
the  formation  of  special  aggressins,  in  the  sense  of  Bail,  is  based  upon 
the  correctness  of  the  supposition  that  the  substances  in  question 
are  in  reality  bodies  sui  generis,  and  this  rests  upon  the  assumption 

1  It  is  noteworthy  that  the  aggressins  by  themselves  are  not  negatively  chemo- 
tactic, but  excite  hyperleukocytosis. 


44     OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

(a)  that  they  are  formed  only  in  the  living  body  of  the  host,  (6)  that 
they  are  not  toxic,  and  (c)  that  the  immunity  which  results  on  injec- 
tion with  aggressin  exudates  is  of  a  type  that  is  definitely  different 
from  the  forms  which  were  known  before,  viz.,  the  antitoxic  and  the 
bacteriolytic  type. 

"Artificial"  Aggressins. — A  careful  investigation  of  Bail's  work 
has  shown  that  these  suppositions  were,  after  all,  not  well  founded. 
Wassermann  and  Citron  have  thus  demonstrated  that  substances 
with  the  identical  properties  of  the  aggressins  of  Bail  can  also  be 
obtained  in  the  test-tube  by  shaking  cultures  of  various  organisms 
(the  swine  plague  and  hog  cholera  bacillus,  for  example)  with  dis- 
tilled water,  proving  that  the  cooperation  of  the  living  organism  of 
the  host  is  not  essential.  The  products  thus  obtained,  in  contra- 
distinction to  Bail's  "natural"  aggressins,  have  been  termed  "artifi- 
cial" aggressins;  there  is  no  real  difference  between  the  two,  however; 
the  quantity  is  smaller,  but  with  the  one  as  with  the  other  it  is  pos- 
sible to  transform  subfatal  doses  of  bacilli  into  fatal  ones  and  to 
bring  about  a  certain  type  of  immunity.  The  second  assumption 
of  Bail  that  aggressin  exudates  are  non-toxic  has  also  been  shown 
to  be  incorrect,  as  the  intraperitoneal  injection  of  sufficiently  large 
amounts  of  dysentery,  cholera,  and  staphylococcus  aggressins,  in 
guinea-pigs,  will  not  only  cause  general  marasmus,  but  actually 
lead  to  the  death  of  the  animal. 

In  fine,  it  has  been  proved  (by  the  precipitin  test,  which  see)  that 
aggressin  exudates  contain  bacillary  proteins,  all  of  which  possess  a 
certain  degree  of  toxic  action,  and  cause  the  formation  of  certain 
antagonistic  substances  when  injected  into  animals.  In  the  light  of 
such  knowledge  it  is  now  possible  to  account  in  a  more  natural  way 
for  those  observations  of  Bail  which  led  him  to  assume  the  existence 
of  aggressins  as  substances  sui  generis.  The  facilitation  of  infection 
is  thus  readily  explained  by  the  fact  that  the  injection  of  the  sub- 
fatal  dose  is  accompanied  by  the  simultaneous  administration  of  a 
certain  amount  of  toxic  material,  and  not  of  a  non-toxic  substance, 
as  Bail  supposed,  so  that  death  is  due  to  the  two  factors  directly  and 
not  to  the  one  indirectly.  This,  however,  was  nothing  new  in  itself, 
since  Bouchard  already  had  shown  that  the  filtrates  of  various 
bacterial  cultures  facilitated  bacterial  infection  (substances  favori- 
santes).  More  recently  Doerr  also  could  prove  that  both  killed 
cultures  of  various  bacteria  and  bacterial  toxins  as  such  (diphtheria 


ARTIFICIAL  AGGRESSINS  45 

and  cholera  toxin)  are  capable  of  producing  a  fatal  effect  when 
injected  together  with  subfatal  doses  of  bacteria. 

It  is  also  quite  clear  now  why  the  simultaneous  injection  of  cer- 
tain bacilli  together  with  suitable  quantities  of  aggressin  exudate 
and  corresponding  bactericidal  (bacteriolytic)  serum  does  not  lead 
to  the  destruction  of  the  bacteria.  Bail  assumed  an  antagonistic 
action  upon  the  bactericidal  substances  on  the  part  of  his  hypo- 
thetical aggressins,  while  the  same  effect  or  rather  lack  of  effect  is 
now  explained  as  the  consequence  of  a  neutralizing  or  inhibiting 
effect  of  normal  bacterial  disintegration  products  (receptors)  upon 
the  bacteriolysins.  As  suitable  treatment  of  animals  with  bacterial 
extracts  and  killed  cultures  of  bacteria  leads  to  the  production  of 
a  certain  type  of  immunity,  in  which  antitoxins  and  certain  bacteri- 
cidal substances  (bacteriolysins)  play  a  prominent  role,  and  as  we 
have  seen  that  bodies  of  that  order  (bacillary  proteins  and  toxins) 
can  actually  be  demonstrated  in  the  aggressin  exudates,  it  follows 
that  there  is  no  ground  for  the  assumption  that  an  antiaggres'sin 
immunity  as  an  immunity  sui  generis  exists. 

A  final  point  which  has  been  raised  against  Bail's  theory  is  the 
fact  that  in  the  antiaggressin  immune  animal  (in  the  sense  of  Bail) 
there  is  no  evidence  either  of  increased  phagocytic  activity  or  of 
increased  resistance  to  the  multiplication  of  bacteria.  Weil,  one 
of  Bail's  pupils,  has  thus  shown  in  chicken  cholera  infection,  for 
example,  that  the  increase  of  bacilli  in  the  immune  animal  may  be 
just  as  intense  as  in  the  control  animal  two  hours  before  death,  while 
the  virulence,  as  tested  on  non-immune  animals,  is  unimpaired,  and 
there  is  no  evidence  of  phagocytosis.  While  Bail's  whole  theory 
of  antiaggressin  immunity  has  thus  fallen  to  the  ground  it  must  be 
admitted  that  in  the  truly  infectious  (septicemic)  diseases,  bacterio- 
lytic immunity  likewise  does  not  play  a  role,  and  the  question  hence 
still  remains  an  open  one;  how  to  account  for  the  undoubted  im- 
munity which  can  be  produced  by  repeated  injection  of  animals 
with  so-called  aggressins.  As  the  protection  of  animals,  which  is 
thus  obtained,  is  not  transferable,  i.  e.,  as  one  animal  cannot  be  ren- 
dered resistant  (immune)  by  the  injection  of  blood  from  an  aggressin- 
immune  animal,  the  question  naturally  suggests  itself,  whether, 
after  all,  we  are  not  dealing  with  a  type  of  immunity  which  is  different 
from  the  other  forms  that  are  commonly  recognized.  This  question 
will  be  discussed  at  greater  length  in  a  later  chapter;  suffice  it  to  say 


46     OFFENSIVE  FORCES  OF  THE  INVADING  MICROORGANISM 

at  this  place  that  there  is  evidence  to  show  that  this  type  of  immunity 
is  essentially  an  antitoxic  immunity,  but  one  in  which  the  antitoxic 
effect  is  probably  the  outcome  of  structural  changes  in  the  chemical 
make-up  of  the  cell  and  not  the  result  of  a  liberation  of  antitoxic 
groups  from  the  cell  and  their  action  upon  toxin  molecules  in  the 
circulation. 

Summary. — To  sum  up:  So  far  as  our  knowledge  of  the  actual 
aggressive  forces  of  the  invading  bacteria  are  concerned  we  must 
admit  that,  barring  the  morphological  changes  with  which  we  have 
become  acquainted,  and  which  we  have  come  to  look  upon  as  pas- 
sively aggressive  forces,  active  forces  furnished  by  the  living  organ- 
isms during  the  infection,  have  not  been  satisfactorily  demonstrated. 
But  we  have  seen  that  bacterial  decomposition  products  in  them- 
selves possess  a  certain  infection-favoring  influence  and  are  in  this 
sense  aggressive.  That  a  decrease  in  the  offensive  forces  of  the  host, 
finally,  is  in  a  measure  equivalent  to  an  increased  aggressivity  of  the 
infecting  bacteria  is  almost  self-evident.  These  forces  will  be  studied 
in  subsequent  chapters,  but  before  entering  upon  their  consideration 
it  may  not  be  out  of  place  to  briefly  review  our  knowledge  of  those 
products  of  bacterial  activity  or  degeneration  which  play  a  role  in 
the  production  of  the  picture  of  the  so-called  infectious  diseases  and 
their  probable  manner  of  action. 


CHAPTER  IV 
BACTERIAL  POISONS 

I  HAVE  pointed  out  in  Chapter  I  that  the  terms  infection  and 
infectious  disease  cannot  be  used  synonymously.  The  existence  of 
an  infectious  disease  itself  implies  the  existence  of  an  infection,  but 
infection  may  exist  in  the  absence  of  any  symptoms  which  denote 
disease.  In  the  ordinary  trypanosomiasis  of  rats,  for  example,  there 
is  nothing  to  suggest  that  the  infected  animal  is  in  any  way  delete- 
riously  affected  by  the  presence  of  the  parasite.  There  is  virtually  a 
symbiosis  between  the  two,  from  which  the  host  does  not  derive 
any  evident  benefit,  to  be  sure,  but  at  the  same  time  it  is  clear  that 
the  trypanosome  on  its  part  does  no  harm. 

In  other  infections,  as  in  anthrax  particularly,  harm  is  actually 
done,  but  the  symptoms  of  harm  appear  so  late  and  are  of  such  brief 
duration  that  one  is  scarcely  warranted  in  speaking  of  the  existence 
of  an  infectious  disease;  when  symptoms  arise  death  is  virtually  at 
hand.  In  such  infections  as  tetanus,  diphtheria,  and  cholera,  on  the 
other  hand,  symptoms  of  disease  become  very  evident  relatively 
early  after  infection,  and  only  too  often  appall  us  through  their  very 
violence. 

On  first  consideration  one  might  imagine  that  the  severe  symp- 
toms in  the  one  group,  and  absence  of  symptoms  in  the  other,  are 
merely  the  expression  of  a  particularly  active  multiplication  of  the 
organisms  in  the  one  as  compared  with  the  other,  and  of  a  corre- 
spondingly severe  intoxication  of  the  macroorganism  with  toxic 
metabolic  products  furnished  by  the  invading  parasite. 

This  explanation,  however,  falls  to  the  ground  if  we  remember 
that  in  the  very  group  in  which  the  most  active  and  generalized 
development  of  organisms  occurs,  symptoms  of  disease  are  virtually 
absent,  while  in  tetanus  and  diphtheria  the  infection  is  essentially 
a  local  one,  and  the  severity  of  the  symptoms  out  of  all  proportion 
to  the  small  number  of  organisms  present.  There  is,  however,  a 
further  important  difference  between  the  two  groups  of  organisms, 


48  BACTERIAL  POISONS 

which  becomes  apparent  at  once  if  we  inject  suitable  animals  with 
killed  cultures  of  the  anthrax  bacillus  on  the  one  hand,  and  the  diph- 
theria and  the  tetanus  bacillus  on  the  other.  It  will  then  be  seen 
that  in  the  anthrax  animal,  as  before,  no  symptoms  develop,  while  in 
the  others  disease  and  death  occur  exactly  as  though  they  had  been 
infected  with  the  living  organisms.  As  the  same  effect  is  obtained, 
if  the  injections  are  made  with  corresponding  cultures  that  have 
been  passed  through  porcelain  filters,  it  is  evident  that  the  dead 
bodies  of  the  bacilli,  as  such,  are  not  concerned  in  the  production  of 
the  result.  This  is  manifestly  due  to  the  presence  of  poisons  in  the 
tetanus  and  diphtheria  filtrates  and  their  absence  in  the  anthrax 
cultures.  The  existence  of  a  clinical  picture  of  tetanus  or  diphtheria 
infection,  in  other  words,  the  development  of  the  corresponding 
infectious  disease,  is  thus  explained,  as  are  also  the  negative  results 
in  anthrax. 

The  question  now  arises:  Are  all  the  so-called  infectious  diseases 
due  to  toxic  substances  derived  from  the  offending  parasites?  This 
question  can,  I  think,  be  answered  in  the  affirmative  for  those  dis- 
eases of  which  the  infecting  agent  is  known.  Regarding  the  nature 
of  the  toxic  agents,  however,  which  are  responsible  for  the  symptom- 
complex  of  the  various  infectious  diseases,  and  the  mechanism  of 
their  action,  our  knowledge  is  as  yet  very  meager. 

Ptomains. — In  the  earlier  days  of  bacteriology,  when  Brieger 
especially  had  shown  in  a  long  series  of  elaborate  investigations 
that  definite  nitrogenous  compounds  of  basic  nature  and  alkaloid- 
like  properties — the  so-called  ptomains — were  formed  from  animal 
matter  in  consequence  of  bacterial  decomposition,  and  that  some  of 
these  bodies  were  poisonous,  hope  ran  high  that  the  application  of 
the  same  methods  to  cultures  of  the  pathogenic  bacteria  proper  would 
lead  to  the  discovery  of  definite  compounds,  to  which  the  symptoms 
of  the  corresponding  diseases  could  be  attributed.  These  hopes 
were,  however,  soon  shattered.  For  a  short  time,  it  is  true,  the 
discovery  of  ptomains,  supposedly  specific  of  various  diseases,  was 
announced  from  different  laboratories.  Brieger  himself  isolated  a 
"typhotoxin"  and  a  "tetanin,"  and  I  well  remember,  when  working 
in  Gautier's  laboratory,  translating  into  French  the  announcement 
from  a  British  source  of  specific  ptomains  for  scarlatina,  measles, 
mumps,  etc.  Later  research  then  showed  that  while  some  ptomains 
are  unquestionably  poisonous  and  may  occasionally  play  a  role  as 


TOXINS  49 

pathogenic  agents,  the  group  as  a  whole  is  of  little  if  any  interest 
from  the  standpoint  of  the  student  of  infection  and  infectious  disease. 

In  the  light  of  more  recent  knowledge  it  is  even  doubtful  whether 
the  serious  symptoms  which  are  observed  in  cases  of  so-called 
ptomain  poisoning  are  in  reality  due  to  ptomains.  Since  we  know 
that  a  specific  organism,  the  bacillus  botulinus,  may  frequently  be 
demonstrated  in  "tainted' '  animal  food  and  that  this  organism  pro- 
duces a  true  toxin — not  a  ptomain — which  is  almost  as  active  as  the 
toxin  of  the  tetanus  bacillus,  one  not  unnaturally  feels  a  little  dubious 
about  the  role  which  the  ptomains  proper  are  supposed  to  play  in 
such  cases. 

The  most  urgent  objection,  however,  which  can  be  raised  against 
the  role  of  the  ptomains  as  active  agents  in  the  causation  of  the  symp- 
toms and  pathological  changes  of  the  infectious  diseases  is,  above  all, 
the  fact  that  they  can  never  reach  such  a  concentration  in  the  living 
body  as  would  suffice  to  bring  about  a  clinical  effect.  In  the  course 
of  Brieger's  typhoid  studies  this  became  especially  manifest,  for 
the  yield  of  his  "typhotoxin"  in  typhoid  cultures,  after  four  weeks' 
incubation,  was  infinitesimally  small  and  often  wanting  altogether. 
Noteworthy,  further,  is  the  fact  that  the  toxic  effect  of  the  isolated 
ptomains  was  always  markedly  less  than  that  of  the  original  culture, 
and  that  pathological  changes  peculiar  to  infections  with  the  corre- 
sponding bacteria  have  never  been  produced  with  ptomains. 

To  sum  up  we  may  say  that  while  ptomains  may  possibly  cause 
disease  or  even  death,  as  in  some  cases  of  cheese  or  meat  poisoning,  or 
in  cases  where  active  absorption  is  taking  place  from  an  abscess  or  a 
gangrenous  focus,  there  is  no  evidence  to  show  that  they  play  a  role 
in  the  pathology  of  the  infectious  diseases  per  se,  and  it  is  doubtful, 
to  say  the  least,  whether  the  effect  which  is  commonly  attributed  to 
them  in  the  conditions  just  mentioned  is  really  the  outcome  of  their 
action. 

Toxins. — If,  then,  the  ptomains  are  eliminated  as  pathogenic 
agents  the  question  arises,  Are  there  any  other  substances  derived 
either  directly  or  indirectly  from  microorganisms  to  the  action  of 
which  the  clinical  picture  of  the  infectious  diseases  could  be  attrib- 
uted? Three  groups  of  substances  are  now  recognized  which  are  of 
moment  in  this  connection,  namely,  the  true  toxins  or  exotoxins,  the 
endotoxins  and  the  bacterial  proteins. 

Of  these  the  endotoxins,  like  the  proteins,  are  part  and  parcel  of 
4 


50  BACTERIAL  POISONS 

the  body  of  the  organisms  and  are  only  liberated  when  these  undergo 
disintegration,  while  the  true  toxins  are  actively  secreted  by  the 
living  cells.  This  is  one  of  the  essential  points  of  difference  also 
which  distinguish  the  exotoxins,  or  toxins  in  short,  as  they  are  usually 
designated  from  the  ptomains.  The  ptomains  are  products  of 
bacterial  action  upon  certain  foodstuffs,  and  their  formation  is,  hence, 
possible  only  when  such  foodstuffs  are  directly  available,  while  toxin 
production  is  within  certain  limitations  independent  of  the  food 
supply,  and  represents  a  specific  function  on  the  part  of  the  micro- 
organisms in  question.  The  toxin  is  in  a  certain  sense  a  product  of 
the  anabolic  activity  of  the  organism,  while  the  ptomain  is  merely 
a  katabolic  product.  The  production  of  a  given  ptomain,  moreover, 
is  not  confined  to  a  given  type  of  organism,  while  true  toxin  produc- 
tion is  specific.  Only  one  organism  is  known  to  form  diphtheria 
toxin,  only  one  is  known  to  produce  tetanus  toxin,  and  only  one 
is  the  source  of  botulismus  toxin.  That  these  toxins  are  actually 
responsible  for  the  clinical  picture  of  the  corresponding  diseases  is 
now  a  recognized  fact,  and  just  as  the  toxins  in  question  are  specific 
products  of  the  bacteria,  so  also  are  the  symptoms  to  which  they 
give  rise  in  a  large  measure  specific  of  the  homologous  infections. 

The  tetanus  toxin  when  injected  by  itself  into  suitable  animals  thus 
causes  tonic  spasms  of  the  muscles  in  the  neighborhood  of  the  point 
of  infection,  increased  reflex  irritability,  dyspnea,  increased  heart 
action,  and  hematolysis  exactly  as  if  the  animal  had  been  inoculated 
with  the  living  bacteria  instead.  The  diphtheria  toxin  produces 
edema,  infiltration,  and  necrosis  at  the  point  of  injection,  increase 
of  temperature  followed  by  a  drop,  and  in  non-fatal  doses  paralysis 
and  marked  emaciation.  Botulismus  toxin,  no  matter  how  intro- 
duced, gives  rise  to  external  and  internal  ophthalmoplegia,  dys- 
phagia,  aphonia,  retention  of  urine  and  feces,  and  to  respiratory 
and  circulatory  disturbances,  with  absence  of  fever  and  of  cerebral 
symptoms,  etc. 

We  may  accordingly  assume  that  the  toxins  in  question  have  a 
special  selective  affinity  for  certain  tissues  and  produce  their  symp- 
toms in  consequence  of  such  affinity.  This  is  seen  especially  well 
in  the  case  of  the  tetanus  toxin,  which  produces  its  clinical  effect 
through  its  action  upon  the  central  nervous  system  to  which  it 
becomes  anchored,  as  is  shown  in  the  following  experiment:  If 
1  gram  of  guinea-pig  brain  is  triturated  with  10  c.c.  of  normal  salt 


TOXINS  51 

solution,  1  c.c.  of  the  resulting  emulsion  is  capable  of  neutralizing  as 
much  as  10  fatal  doses  of  the  toxin  (i.  e.,  fatal  for  white  mice)  and  of 
causing  a  marked  decrease  in  the  toxic  action  of  as  much  as  60  fatal 
doses  of  the  toxin.  The  blood,  liver,  kidneys,  spleen,  and  muscles,  on 
the  other  hand,  do  not  possess  this  neutralizing  power.  The  affinity 
which  exists  between  the  hemolytic  toxin  (staphylolysin)  produced 
by  staphylococcus  aureus  and  red  corpuscles  is  similarly  shown  when 
the  toxin  is  allowed  to  act  upon  the  red  cells  at  0°  C.,  at  which  tem- 
perature hemolysis  does  not  take  place;  if,  then,  the  corpuscles  are 
thrown  down  by  centrifugation  the  supernatant  fluid  will  be  found 
to  have  lost  its  hemolytic  action,  while  the  red  cells  have  taken  up 
the  active  principle  and  hold  this  so  tenaciously  that  it  cannot  be 
abstracted  again,  even  on  repeated  washing  with  normal  salt  solu- 
tion. Other  cells  are  practically  inert  in  this  respect.  Another  toxin 
produced  by  the  staphylococcus — the  leukocydin — has  a  similar 
selective  affinity  for  leukocytes. 

The  activity  of  the  toxins  is  most  remarkable  and  far  exceeds 
that  of  the  most  toxic  ptomains.  One  preparation  of  tetanus  toxin 
could  thus  be  shown  to  be  fatal  for  mice  in  a  dose  of  0.00000025 
gram,  and  another  in  the  still  smaller  dose  of  0.00000005  gram.  A 
culture  of  the  bacillus  botulinus  similarly  produced  a  fatal  effect 
in  doses  varying  between  0.01  and  0.00005  c.c.  These  figures  assume 
increased  significance  if  we  remember  that  the  toxins  have  never  been 
prepared  in  a  state  of  chemical  purity  and  that  our  purest  products 
are  still  contaminated  with  a  preponderating  amount  of  inert 
material. 

While  the  diphtheria  bacillus,  the  tetanus  bacillus,  and  the  bacillus 
botulinus  are  usually  mentioned  as  being  the  only  bacteria  which 
secrete  a  soluble  toxin,  it  is  now  known  that  a  number  of  other 
organisms  also  furnish  soluble  toxins,  and  there  is  hence  good  ground 
for  the  belief  that  some  of  the  symptoms  which  are  observed  in  the 
corresponding  infections  may  be  referable  to  such  toxins  and  are  in  a 
measure  characteristic.  The  organisms  in  question  are  the  dysentery 
bacillus,  the  bacillus  of  symptomatic  anthrax,  the  cholera  vibrio  and 
closely  related  organisms  (vibrio  El  Tor,  vibrio  Nasik),  the  typhoid 
bacillus,  the  pyocyaneus,  and  the  staphylococcus  aureus.  Of  these 
the  dysentery  toxin  (in  the  animal  experiment)  produces  paralyses— 
(especially  of  the  posterior  extremities),  hemorrhagic  diarrhea  and 
subnormal  temperature;  the  typhoid  toxin — diarrhea,  hyperemia, 


52  BACTERIAL  POISONS 

and  hemorrhages  from  the  intestinal  mucosa;  the  staphylococcus 
toxin  causes  hard  infiltration  and  necrosis  at  the  point  of  injection, 
besides  renal  lesions,  hemolysis,  and  leukocytolysis;  the  toxin  of 
the  cholera  vibrio,  drop  of  temperature,  pareses,  rectal  prolapse, 
and  death  after  five  hours  or  later. 

Endotoxins. — While  the  action  of  the  true  toxins  is  thus  individ- 
ually fairly  specific,  so  that  one  can  speak  of  a  hematoxin,  a  neuro- 
toxin,  a  leukocytotoxin,  etc.,  or  as  would  probably  be  more  correct: 
of  a  hematoxic  or  a  neuro toxic  component  of  a  toxin  group,  this  is 
in  a  measure,  though  possibly  to  a  less  extent,  also  true  of  the  endo- 
toxins,  which,  as  already  explained,  are  not  secreted  by  the  living 
organisms,  but  are  only  set  free  after  the  death  and  disintegration 
of  the  parasites.  Whether  this  latter  feature  is  in  reality  sufficient 
to  warrant  such  a  complete  separation  of  the  endo-  from  the  exotoxins 
may  be  questioned,  particularly  since  the  principal  additional  differ- 
ential factor,  viz.,  the  inability  of  the  endotoxins  to  give  rise  to  anti- 
toxin formation  on  injection  into  suitable  animals,  is  in  the  light  of 
recent  work  no  longer  recognized.  For  practical  purposes,  however, 
the  separation  of  the  endotoxins  as  a  class  is,  at  the  present  time  at 
least,  convenient. 

In  the  earlier  days  of  bacteriology  the  existence  of  the  endo- 
toxins as  substances  sui  generis  had  been  overlooked,  and  their  effect 
attributed  to  the  action  of  the  bacterial  proteins  which  themselves 
are  toxic  to  a  greater  or  less  degree.  Their  independent  character  is, 
however,  now  assured  by  the  fact  that  their  injection  into  suitable 
animals  gives  rise  to  the  production  of  antitoxins,  which  are  capable 
of  neutralizing  the  corresponding  toxins,  and  that  the  toxic  effect 
rapidly  diminishes  on  keeping  and  is  seriously  impaired  by  exposure 
to  higher  temperatures  (55°  to  60°),  while  the  proteins  resist  a  tem- 
perature of  120°  C.  for  a  whole  hour.  Their  action  on  the  living 
animal,  moreover,  is  totally  different  from  that  of  the  bacterial 
proteins. 

Bacterial  Proteins. — The  bacterial  proteins  are  essentially  pyo- 
genic  in  character,  which  property,  according  to  Buchner,  is  com- 
mon to  most  if  not  to  all  the  bacteria.  It  has  been  demonstrated 
for  the  staphylococcus,  streptococcus,  Friedlander's  pneumo- 
bacillus,  the  bacillus  coli  communis,  acidi  lactici,  proteus,  prodigiosus, 
cyanogenes,  subtilis,  the  sarcina  aurantiaca,  the  vibrio  of  Finkler- 
Prior,  certain  water  bacteria,  etc.  In  some  of  the  organisms  the 


SUMMARY  53 

pyogenic  action  does  not  manifest  itself,  because  death  results 
too  early;  but  it  can  be  demonstrated,  nevertheless,  if  the  same 
organism  be  tested  in  less  resistant  animals.  While  the  chicken 
cholera  bacillus  thus  kills  chickens  without  evidence  of  pyogenic 
action,  the  injection  of  sheep,  horses,  or  guinea-pigs  leads  to  the 
formation  of  abscesses  at  the  points  of  injection  without  a  generalized 
septicemia.  This  observation  in  itself  goes  to  show  that  the  specific- 
ally toxic  effect  of  the  organisms  in  question  is  something  separate 
and  apart  from  the  pyogenic  effect  and  evidently  due  to  separate 
substances. 

Aside  from  their  general  and  non-specific  pyogenic  properties  the 
bacterial  proteins  in  themselves  are  not  markedly  dangerous  to  the 
injected  animal,  but  they  have  gained  new  importance,  since  it  has 
been  demonstrated  that  the  introduction  of  foreign  albumins,  of 
whatever  kind,  leads  not  to  increased  resistance  (immunity)  against 
such  proteins,  but,  on  the  contrary,  to  hypersensitiveness  (anaphyl- 
axis,  allergia),  such  that  a  subsequent  injection,  after  a  certain 
interval  of  time,  may  produce  the  most  serious  symptoms  and  even 
death.  As  a  sensitiveness  of  this  order  can  very  well  be  imagined  to 
occur  in  the  course  of  a  bacterial  disease,  the  thought  has  naturally 
suggested  itself  that  certain  symptoms  occurring  during  the  later 
stages  of  various  infections  may  be  explained  upon  this  basis  (see 
section  on  Anaphylaxis) .  But  even  disregarding  their  possible 
significance  from  this  point  of  view,  their  pyogenic  property  in 
itself  is  sufficient  to  render  them  important.  Through  their  attract- 
ing effect  upon  the  leukocytes  (positive  chemotaxis)  they  immediately 
assume  a  clinical  interest,  and  in  certain  infections  no  doubt  (staphy- 
lococcus,  streptococcus,  colon  bacillus)  they  are  responsible  for  a  large 
portion  of  the  clinical  picture  (anemia,  hyperleukocytosis,  pus 
formation,  fever). 

Summary. — To  sum  up  then  we  have  seen  that  the  picture  of  the 
infectious  disease,  insofar  as  the  microorganisms  themselves  are 
concerned,  may  be  referable  (a)  to  the  action  of  special  exotoxins 
which  are  actively  secreted  by  the  living  bacteria;  (6)  to  the  action 
of  somewhat  less  specific  endotoxins  which  enter  into  play  only  after 
the  death  and  destruction  of  the  organisms;  and  (c)  to  the  relatively 
non-specific  action  of  the  bacterial  proteins.  The  mechanism  of  the 
action  of  these  various  substances  will  be  considered  in  some  detail 
in  a  subsequent  chapter.  At  this  place  it  will  suffice  to  point  out 


54  BACTERIAL  POISONS 

that  insofar  as  a  direct  chemical  effect  upon  the  cells  of  the  body 
is  concerned,  this  can  only  take  place  if  a  mutual  affinity  exists 
between  such  cells  and  the  toxic  substances  or  their 'derivatives. 
But  it  does  not  follow  that  because  of  such  a  mutual  affinity  a 
toxic  effect  must  of  necessity  result.  This  can  only  occur  if  the 
combination  with  the  toxic  principle  implies  a  toxic  effect.  If,  then, 
we  observe  a  toxic  effect  clinically,  upon  the  central  nervous  system, 
for  example,  this  does  not  necessitate  the  conclusion  that  the  toxin 
does  not  act  upon  other  structures  of  the  body  also,  but  clinically 
the  toxic  effect  is,  of  course,  the  only  effect  which  excites  our  attention. 

Remembering  the  curious  interaction  between  different  organs, 
however,  the  thought  naturally  suggests  itself  that  the  toxic  bacterial 
products  might  exert  a  toxic  effect  not  only  directly  but  also  indi- 
rectly. This  possibility  has  apparently  not  received  the  attention 
which  it  deserves,  but  must  nevertheless  be  borne  in  mind.  It  is 
perfectly  conceivable  that  a  toxin  might  act  upon  a  certain  organ 
in  a  way  to  impair  its  function,  without  actually  endangering  the 
integrity  of  the  cells  as  such,  but  that  the  impairment  of  its  function 
may  carry  in  its  trail  secondary  effects  which  become  apparent  to 
the  clinician  at  once,  while  the  primary  action  escapes  attention. 
Every  physician  is  familiar  with  the  effect  of  various  infections  upon 
the  gastro-intestinal  functions,  on  the  occurrence  of  constipation 
defective  secretion  of  hydrochloric  acid,  etc.,  factors  which  we  now 
know  to  be  controlled  to  a  large  extent,  if  not  entirely,  by  hormone 
action,  and  it  is  clear  in  view  of  the  interdependence  of  the  gastro- 
intestinal hormones,  that  interference  at  any  one  point  in  the  chain 
might  readily  upset  the  digestive  equilibrium  and  lead  to  various 
further  disturbances  of  the  metabolism. 

Then,  again,  as  I  have  already  pointed  out,  there  is  a.  certain  danger 
from  the  action  of  those  very  products  (antibodies)  which  the  body 
itself  forms,  primarily  no  doubt  as  a  defensive  reaction,  against  the 
products  derived  from  the  bacteria  and  of  which  more  will  be  said 
in  later  chapters.  It  is  clear  at  any  rate  that  the  picture  of  the  infec- 
tious disease  is  unquestionably  the  composite  of  more  factors  than 
we  are  apt  to  think  on  first  consideration,  and  some  of  which  no 
doubt  will  be  found  to  explain  such  symptoms  as  the  mysterious 
death  from  anthrax,  for  example,  where  evidences  of  actual  disease 
are  wanting  until  the  end  is  near,  and  where  the  first  symptoms  are 
practically  the  last  ones.  To  explain  this  point,  undue  prominence 


INFECTIONS  WITH  ANIMAL  PARASITES  55 

has  been  given  in  the  past  to  mechanical  factors.  It  was  suggested 
that  the  occlusion  of  extensive  capillary  districts  with  densely  matted 
masses  of  anthrax  bacilli,  or,  as  in  some  of  the  severest  types  of 
tropical  malaria,  with  masses  of  plasmodia,  was  directly  responsible 
for  the  fatal  issue.  This  purely  mechanical  element  cannot,  of  course, 
be  ignored,  but  in  the  light  of  our  present  knowledge  of  the  physio- 
logical pathology  of  the  infectious  diseases,  we  are  warranted  in  the 
belief  that  the  future  will  bring  a  more  satisfactory  explanation. 

Infections  with  Animal  Parasites. — While  the  foregoing  considerations 
apply  more  particularly  to  bacterial  infections,  similar  conditions  no 
doubt  exist  in  infections  with  animal  parasites.  Primary  infection 
is  here  often  facilitated  by  the  intervention  of  special  infection  car- 
riers. We  thus  know  that  malaria  is  transmitted  through  the  bite 
of  infected  mosquitoes  (Anopheles  maculipennis),  trypanosomiasis 
through  biting  flies  (Glossina  fusca  and  tachinoides),  African  relaps- 
ing fever  and  Texas  cattle  fever  through  certain  ticks  (Ornithodorus 
moubata  and  Boophilus  bovis  respectively. 

With  other  organisms,  such  as  the  Treponema  pallidum  (Spiro- 
chete)  we  may  assume  the  existence  of  tiny  breaks  in  the  continuity 
of  the  epithelial  covering,  as  in  the  majority  of  the  bacterial  infec- 
tions, while  with  still  others,  like  the  ameba  coli,  we  may  imagine 
that  the  epithelial  lining  is  first  destroyed  by  the  parasite  itself. 
What,  then,  happens,  after  actual  invasion  of  the  deeper  structures 
has  taken  place,  we  can  only  surmise,  but  it  would  appear  that  the 
aggressivity  of  the  animal  parasites  is  upon  the  whole  even  greater 
than  that  of  the  bacteria.  A  more  or  less  extensive  infection  appar- 
ently occurs  in  all  cases,  in  which  the  microorganism  has  once  gained 
a  foothold,  some  of  the  organisms  in  question  multiplying  in  the  blood 
stream  (malaria,  trypanosomiasis,  relapsing  fever),  others  in  the 
tissues  (syphilis,  amebiasis),  only  too  often  without  much  show  of 
active  resistance  on  the  part  of  the  host.  What  the  aggressive  forces 
are  which  the  animal  parasite  has  at  its  disposal  we  do  not  know. 
In  the  case  of  the  malarial  parasite  these  are  manifestly  directed  with 
a  remarkable  degree  of  specificity  against  the  red  corpuscles.  Having 
once  gained  an  entrance  they  are  evidently  perfectly  secure;  appar- 
ently they  are  open  to  attack  only  while  they  exist  free  in  the  plasma. 

Of  the  formation  of  toxic  products  on  the  part  of  the  animal  para- 
sites nothing  definite  is  known.  The  clinical  history,  however,  would 
suggest  this.  In  malaria  the  occurrence  of  the  chill  and  fever  and 


56  BACTERIAL  POISONS 

the  lack  of  relation,  which  exists  between  the  degree  of  anemia  and 
the  number  of  the  parasites,  could  hardly  be  accounted  for  in  any 
other  way,  while  the  mere  destruction  of  the  red  cells  itself  could  be 
explained  by  the  manifest  proteolytic  activity  of  the  parasite  and 
consequent  changes  in  the  osmotic  tension  in  the  cell.  In  trypanoso- 
miasis  the  late  symptoms  at  least  (sleeping  sickness)  would  suggest 
a  toxic  cause,  and  in  relapsing  fever  the  entire  symptom  complex 
is  toxic  in  character.  As  I  have  said,  however,  we  have  no  definite 
knowledge  on  the  subject,  which  is,  no  doubt,  owing  to  the  fact  that 
until  quite  recently  we  were  unable  to  cultivate  the  parasites  in 
question  as  we  would  bacteria,  and  could  hence  not  study  the 
products  of  either  their  growth  or  disintegration. 


CHAPTER  V 
THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

IN  the  foregoing  chapter  we  have  briefly  reviewed  the  aggressive 
forces  of  the  bacteria  and  the  manner  in  which  they  bring  about  some 
of  the  symptoms  of  the  infectious  diseases,  while  we  have  said  nothing 
as  yet  of  the  mechanism  by  which  the  macro  organism  defends  itself 
against  the  infection  per  se,  and  the  action  of  those  poisonous  prod- 
ucts which  are  so  largely  responsible  for  the  clinical  picture  of  the 
infections.  This  will  be  our  special  problem  in  the  chapters  which 
are  now  to  follow.  We  may  here  distinguish  between  those  forces 
which  are  at  the  disposal  of  the  animal  body  at  the  moment  of  infec- 
tion and  those  which  develop  only  in  the  course  of  the  infection, 
and  because  of  the  infection.  The  former  comprise  the  phagocytic 
forces  of  the  body  cells  and  the  normal  bactericidal  power  of  the 
serum,  while  the  second  class  includes  the  various  antibodies,  so- 
called,  viz.,  those  substances  which  are  liberated  from  the  cells  in 
consequence  of  the  introduction  into  the  circulation  of  cells  or  cell 
products  which  are  foreign  to  the  body.  In  addition  we  recognize 
still  other  defensive  factors,  which  in  a  measure  are  operative  in  a 
passive  way,  but  which  are  nevertheless  of  great  importance  from  the 
standpoint  of  immunity. 

PHAGOCYTOSIS 

While  in  the  lowest  forms  of  animal  life  phagocytosis  is  a  sine  qua 
non  for  the  very  existence  of  the  individual,  representing  as  it  does 
the  only  mechanism  by  which  the  animal  is  capable  of  apprehending 
its  food,  insofar  at  least  as  this  is  of  an  organized  type,  this  property 
is  lost  to  a  greater  or  less  extent  as  a  common  cellular  characteristic 
in  the  higher  forms,  but  is  retained  by  certain  cells  in  all  forms  of 
animal  life  from  the  lowest  invertebrate  to  the  highest  vertebrate. 
In  the  latter  the  phagocytic  function  is,  generally  speaking,  confined 
to  cells  which  are  derivatives  of  the  original  mesoderm,  the  nerve 
cell  being  the  only  apparent  exception,  on  the  basis  at  least  that 


58        THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

leprosy  bacilli  in  variable  number  have  been  encountered  in  these 
cells,  and  assuming  that  their  entrance  occurred  through  the  activity 
of  the  nerve  cell  itself.  All  other  cells  in  which  phagocytosis  has 
been  observed  are  mesodermal  derivatives. 

Microphages  and  Macrophages. — Metschnikoff,  to  whom  we  are 
indebted  for  so  much  of  our  knowledge  of  phagocytosis,  in  all  its 
aspects,  divides  the  cells  which  are  endowed  with  this  power  into  two 
large  groups,  viz.,  the  microphages  and  macrophages.  The  former 
group  is  represented  practically  exclusively  by  the  neutrophilic 
polymorphonuclear  and  polynuclear  leukocytes,  while  the  mast  cells 
and  eosinophiles  either  do  not  engage  in  phagocytosis  at  all,  or  do  so 
only  to  a  slight  and  unimportant  extent.  The  macrophages,  on  the 
other  hand,  comprise  the  large  mononuclear  leukocytes  of  the  blood, 
the  endothelial  cells  lining  the  peritoneal  cavity,  the  sessile  (fixed) 
mononuclear  cells  of  the  splenic  follicles  and  the  sinuses  of  the  lymph 
glands,  the  stellate  cells  of  Kupffer  in  the  liver,  the  large  mononu- 
clear, so-called  alveolar,  epithelial  cells  of  the  lungs,  which  latter  two 
according  to  Metschnikoff  are  in  reality  large  mononuclear  leuko- 
cytes; and  finally  the  bone  corpuscles  and  the  myeloplaxes  or  giant 
cells  of  the  bone  marrow. 

Phagocytic  Function  of  Various  Types  of  Cells. — What  the  significance 
of  the  phagocytic  function  of  these  various  types  of  cells  really  is  in 
an  organism  in  which  so  extensive  a  differentiation  has  taken  place 
as  in  the  vertebrate  animal  is  largely  a  subject  of  conjecture.  We 
may  imagine,  however,  that  under  normal  conditions  phagocytosis 
plays  no  essential  role,  and  merely  represents  a  general  property  of 
mesoblastic  protoplasm  which  is  of  interest  ontogenetically,  but 
of  no  practical  importance.  But  we  can  readily  see  that  this  same 
function,  even  though  it  remains  dormant,  while  the  body  is  in 
perfect  health,  immediately  assumes  importance  of  the  first  order, 
if  foreign  cells  are  introduced  from  without.  Under  such  conditions 
the  phagocyte  is  placed  in  a  similar  position  as  its  ancestral  proto- 
type, the  ameba,  and  it  would  accordingly  display  the  same  or 
similar  functions,  of  which  the  phagocytic  action  is  one.  In  the 
case  of  the  microphages  (polynuclear  neutrophilic  leukocytes)  at 
any  rate  we  have  no  evidence  that  their  phagocytic  power  enters 
into  action  under  normal  conditions,  while  with  the  macrophages 
the  disposal  of  morphological  products  of  normal  cell  degeneration 
may  possibly  be  a  normal  office  of  the  cells  in  question. 


PHAGOCYTIC  FUNCTION  OF  VARIOUS  TYPES  OF  CELLS     59 

Both  types,  however,  are  capable  of  taking  up  foreign  cells  when 
these  are  introduced  from  without,  although  it  lies  in  the  nature  of 
their  differing  mobility  (the  granular  leukocytes  being  essentially 
mobile  and  the  macrophages  sessile)  that  the  former  play  a  more 
important  role  in  the  actual  conflict  between  the  invading  cells  and 
the  defensive  forces  of  the  host.  That  the  polynuclear  neutrophilic 
leukocytes  more  especially  will  take  up  bacteria  and  protozoa1  has 
been  known  for  many  years,  and  has  been  demonstrated  not  only 
in  vitro,  but  manifestly  occurs  also  in  the  living  body,  as  is  sug- 
gested by  the  findings  in  gonorrheal  pus,  in  the  cerebrospinal 
exudate  of  epidemic  cerebrospinal  meningitis,  in  the  peritoneal  fluid 
of  general  peritonitis,  etc.,  where  many  of  the  offending  organisms 
may  be  found  enclosed  in  leukocytes. 

The  enormous  extent  to  which  phagocytosis  of  bacteria  may  go 
is  well  illustrated  by  the  following  example  which  came  under  my 
observation  a  few  years  ago.  In  a  patient  who  was  dying  from 
epidemic  cerebrospinal  meningitis  we  could  demonstrate  the  presence 
of  meningococci  directly  in  the  stained  preparation  and  calculated 
their  actual  number  to  be  7,380,000  per  cubic  centimeter.  The  vast 
majority  of  these  were  enclosed  in  polynuclear  neutrophiles  and  in 
large  mononuclear  cells  which  I  was  inclined  to  view  as  endothelial  cells. 

The  value  of  such  forces,  if  they  are  actually  directed  against 
living  pathogenic  organisms  in  the  infected  body,  is,  of  course,  self- 
evident.  In  the  earlier  days  of  our  knowledge  of  phagocytosis,  when 
Metschnikoff  for  the  first  time  insisted  upon  the  importance  of  the 
process  from  the  standpoint  of  immunity,  it  was  argued  that  the 
leukocytes  were,  after  all,  mere  scavengers  and  could  only  take  up 
organisms  that  had  already  been  killed  by  the  bactericidal  substances 
of  the  serum  (see  the  following  chapter),  and  that  their  value  as  a 
defensive  force  was  thus  merely  secondary.  Metschnikoff  and  his 
pupils,  however,  have  demonstrated  in  a  series  of  investigations  that 
the  leukocytes  can  actually  take  up  living  and  virulent  bacteria  in 
the  living  host.  They  showed  this  for  the  first  time  in  anthrax- 
immune  pigeons  where  they  were  able  to  recover  anthrax  bacilli 
from  the  leukocytes  of  the  peritoneal  exudate,  both  by  culture  and 
animal  inoculation. 

1  While  this  is  true,  generally  speaking,  it  should  be  remembered  that  the 
phagocytic  action  of  the  microphages  is  essentially  directed  against  bacteria, 
and  that  of  the  macrophages  against  animal  organisms. 


60   THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

Special  stress  is  laid  upon  the  fact  that  these  results  were  obtained 
in  immune  animals,  as  it  could  only  be  shown  in  this  way  that  the 
leukocytes  are  capable  of  taking  up  not  only  living  but  also  virulent 
bacteria,  i.  e.,  bacteria  which  in  the  non-immune  organism  would 
have  produced  a  general  infection. 

That  living  foreign  cells  are  subject  to  phagocytosis  is  also  well 
shown  by  the  following  experiments :  If  a  guinea-pig  is  injected  intra- 
peritoneally  with  goose's  blood  containing  the  spirillum  of  Sacharoff, 
which  produces  a  septicemic  infection  in  geese,  and  if  a  drop  of  the 
exudate  is  then  examined  under  the  microscope,  phagocytosis  of  the 
spirilla — in  this  case  by  macrophages — can  be  observed  directly,  and 
it  will  be  seen  that  many  of  the  organisms  are  quite  motile  yet  with 
their  free  ends,  while  the  remainder  of  the  parasites  has  already 
been  taken  up  by  the  cells. 

Destruction  of  Bacteria  by  Phagocytosis. — While  there  can  thus  be 
no  doubt  that  both  micropbages  and  macrophages  can  take  up  living 
foreign  cells  the  next  question  of  importance  is,  What  happens  to  the 
organisms  after  they  have  been  taken  up?  Two  possibilities,  of  course, 
suggest  themselves.  We  may  imagine,  on  the  one  hand,  that  the 
phagocyte  destroys  the  bacteria,  and  a  priori  this  would  seem  the 
most  natural  thing  to  expect.  On  the  other  hand  the  possibility  at 
least  must  be  borne  in  mind  that  the  bacteria  may  destroy  the 
phagocytes.  If  this  were  to  happen  we  could  readily  understand 
that  phagocytosis  might  at  times  be  of  some  danger  to  the  animal, 
for  we  could  see  that  a  chance  might  thus  be  afforded  for  a  wider 
distribution  of  the  parasites.  The  possibility  of  such  an  occurrence 
is  suggested  by  the  fact  that  the  phagocytosis  of  tubercle  bacilli  by 
giant  cells,  for  example,  usually  leads  to  the  destruction  of  the  latter 
and  not  necessarily  to  the  death  of  the  bacilli.  It  must  be  admitted, 
however,  that  the  greater  weight  of  the  evidence  goes  to  show  that 
sooner  or  later  the  ingested  organisms  are  killed.  The  intracellular 
granular  degeneration  of  bacteria  which  one  can  observe  directly 
under  the  microscope  certainly  points  in  that  direction. 

Of  the  manner  in  which  the  destruction  of  the  bacteria  is  brought 
about  we  are  as  yet  in  comparative  ignorance.  Recent  research 
seems  to  show  that  the  leukocytes  contain  special  endolysins  which 
may  be  operative  in  this  direction.  This  is  really  what  one  would 
expect,  remembering  that  the  proteolytic  enzymes  of  the  cell  can 
hardly  exercise  any  germicidal  action,  and  that  in  many  of  the  lower 


BACTERIOTROPINS  61 

forms  of  animal  life  there  is  direct  evidence  of  a  destruction  of  the 
captured  living  cell  preparatory  to  its  digestion.  Much  work, 
however,  remains  to  be  done  in  this  direction. 

While  leukocytes  are  capable  of  taking  up  certain  bacteria  in  the 
absence  of  blood  serum — spontaneous  phagocytosis — the  majority 
of  those  organisms  which  are  pathogenic  for  man  and  the  higher 
animals  become  subject  to  phagocytic  action  to  a  notable  degree, 
only  after  they  have  been  exposed  to  the  action  of  fresh  serum.  This 
fact  was  emphasized  already  by  Denys  and  Leclef,  who  noted  that 
phagocytosis  was  greatly  facilitated  if  the  process  were  permitted  to 
take  place  in  the  presence  of  serum  from  an  animal  that  had  been 
immunized  against  the  corresponding  organism.  In  contradistinc- 
tion to  Metschnikoff,  who  referred  this  peculiar  effect  to  the  possible 
presence  in  the  serum  of  substances  which  exercised  a  stimulating 
action  upon  the  activity  of  the  leukocytes  (stimulins),  Denys  and 
Leclef  suggested  that  the  effect  of  the  serum  might  be  directed  against 
the  bacteria  in  the  sense  that  the  exo-  and  endotoxins  of  the  latter 
were  neutralized  and  the  organisms  thus  deprived  of  their  most 
active  defensive  weapon  against  the  phagocytic  activity  of  the 
leukocytes. 

Opsonins. — Wright  and  Douglas  then  proved  that  these  substances 
which  they  could  demonstrate  in  normal  serum  also,  actually  prepare 
the  bacteria  for  phagocytosis.  This  was  shown  by  suspending 
organisms  for  a  while  in  fresh  serum,  washing  them  with  normal 
salt  solution  and  then  exposing  them  to  the  action  of  leukocytes, 
when  phagocytosis  promptly  occurred,  while  similar  exposure  of  the 
leukocytes  to  serum  and  subsequent  washing  gave  rise  to  negative 
results.  Wright  and  Douglas  hence  termed  the  substances  in  question 
opsonins  (from  the  Latin  verb  opsonare,  to  cater  to,  to  prepare 
pabulum  for),  and  expressed  the  opinion  that  the  opsonins  of  normal 
serum  and  immune  serum  are  identical. 

Bacteriotropins. — This,  however,  is  denied  by  others,  such  as  Neuf  eld 
and  Rimpau,  who  confirmed  the  findings  of  Denys  and  his  pupils 
regarding  the  presence  of  prophagocytic  substances  in  immune 
serum  and  named  these  bodies  bacteriotropins,  the  essential  basis  for 
their  belief  in  the  difference  of  the  two  groups  of  substances,  at  the 
time  being  the  relative  thermostability  of  the  bodies  found  in  the 
immune  serum,  as  contrasted  with  the  instability  of  the  opsonins  of 
normal  serum  when  exposed  to  a  temperature  of  only  56°  C.  for 


62        THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

thirty  minutes.  Subsequent  investigations  by  numerous  observers 
have  furnished  additional  support  to  Neuf eld's  view,  the  non- 
specificity  of  the  normal  opsonins  (established  by  myself  and 
Lamar  as  well  as  by  Neufeld,  Levaditi  and  Inman,  Ritchie, 
Russell,  and  others)  being  one  of  the  most  weighty  arguments  in 
its  favor. 

This  is  also  shown  by  the  observation  that  the  normal  opsonins 
are  complex  substances,  phagocytic  action  depending  upon  the 
joined  action  of  two  bodies,  viz.,  a  thermolabile  component  (opsonic 
complement)  and  a  second  component  (opsonic  amboceptor)  which 
unites  with  the  first  mentioned,  on  the  one  hand,  and  the  bacterium, 
on  the  other,  whereas  the  bacteriotropins  of  immune  sera  can  act 
independently  (of  complement).  This  difference  is  shown  still 
further  by  the  different  effect  which  normal  and  immune  sera  exercise 
upon  phagocytosis,  when  virulent  as  compared  with  non-virulent 
organisms  are  studied  in  this  direction;  for,  whereas  non- virulent 
strains  of  staphylococci,  streptococci,  pneumococci  and  anthrax 
bacilli  for  example,  readily  succumb  to  phagocytosis  in  the  presence 
of  fresh  normal  serum,  highly  virulent  forms  do  so  only  in  the  pres- 
ence of  immune  serum. 

Susceptibility  to  Opsonification. — In  this  connection  it  is  interesting 
to  note  that  even  aside  from  the  degree  of  virulence,  marked  differ- 
ences exist  in  the  susceptibility  to  opsonification  on  the  part  of 
different  organisms.  Gruber  and  Futaki  have  thus  established  three 
groups  in  reference  to  their  behavior  toward  active  and  inactive 
normal  serum. 

GROUP  I.  Bacteria  which  are  readily  taken  up  by  leukocytes  in 
the  presence  of  fresh  serum,  but  not  in  the  presence  of  inactivated 
(heated)  serum. 

Staphylococcus  pyogenes  aureus. 

Streptococcus  pyogenes. 

Diplococcus  pneumonise. 

Bacterium  coli. 

Bacillus  prodigiosus  suum. 

Bacillus  subtilis. 

Bacillus  erysipelatosus. 

Vibrio  proteus. 

Bacillus  diphtherise. 


ROLE  OF  LEUKOCYTES  IN  PHAGOCYTOSIS  63 

GROUP  II.  Bacteria  which  are  readily  taken  up  by  leukocytes 
in  the  presence  of  fresh  serum,  but  to  a  slight  extent  also  in  the 
presence  of  inactivated  (heated)  serum. 

Bacillus  pyocyaneus. 

Bacillus  sui  pestifer. 

Bacterium  sui  septicum. 

GROUP  III.  Bacteria  which  are  not  susceptible  to  opsonic  action, 
and  which  are  hence  taken  up  by  leukocytes  equally  well  in  the 
presence  of  active  as  well  as  of  inactive  serum. 

Virulent  chicken  cholera  bacilli. 

Virulent  Asiatic  cholera  bacilli. 

Wright  and  Douglas  give  the  following  list  of  organisms  as  being 
subject  to  opsonification : 

Staphylococcus  aureus  and  albus. 

Bacillus  pestis. 

Micrococcus  melitensis. 

Diplococcus  pneumonise. 

Bacterium  coli. 

Bacillus  dysenterise  (Shiga). 

Bacillus  anthracis. 

Bacillus  typhosus. 

Vibrio  cholerse. 

Bacillus  tuberculosis. 

The  diphtheria  bacillus  (in  contradistinction  to  Gruber  and 
Futaki)  and  the  bacillus  xerosis  they  found  to  be  uninfluenced  by 
the  opsonins  of  normal  serum,  phagocytosis  actually  progressing 
more  readily  in  inactive  than  in  fresh  serum. 

Role  of  Leukocytes  in  Phagocytosis. — Whether  or  not  the  leukocytes 
play  an  absolutely  indifferent  role  in  phagocytosis,  uninfluenced  by 
constituents  of  the  serum,  still  remains  an  open  question.  The  fact 
that  the  leukocytes  of  a  given  animal  will  take  up  organisms  which 
have  been  subjected  to  the  action  of  the  serum  not  only  of  animals 
of  different  species  and  genera,  but  of  different  classes  of  animals, 
would  on  first  thought  suggest  this.  But,  on  the  other  hand,  it  has 
been  found  that  leukocytes,  from  different  animals,  show  a  different 
phagocytic  activity  toward  organisms  that  have  been  opsonified  by 
one  given  serum.  Rosenow  could  show  that  in  pneumonia  the 
patient's  own  leukocytes  are  more  actively  phagocytic  than  normal 


64       THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

ones,  and  that  this  difference  is  independent  of  the  action  of  the 
serum.  As  the  leukocytes  from  other  infectious  diseases  (appendi- 
citis and  puerperal  sepsis)  showed  a  similar  behavior  toward  pneumo- 
cocci,  Rosenow  concluded  that  this  difference  is  essentially  the 
expression  of  an  increased  power  of  resistance  and  higher  activity 
on  the  part  of  the  younger  cells  which  find  their  way  into  the  circu- 
lation in  acute  septic  processes,  and  which  in  normal  blood  are  in  the 
minority.  Differences  of  this  order  must  unquestionably  exist,  but 
they  have  after  all  but  little  to  do  with  the  question  whether  the 
leukocytes  as  phagocytes  play  only  a  secondary  role  in  the  defence 
of  the  infected  organism  against  the  invading  bacteria.  The  greater 
part  of  the  evidence  is  certainly  in  favor  of  this  view;  the  existence 
of  substances  which  directly  influence  leukocytic  action  (stimulins, 
in  the  sense  of  Metschnikoff)  has  not  at  any  rate  been  satisfactorily 
demonstrated. 

Effect  of  Opsonification  on  Bacteria. — Of  the  manner  in  which  opsoni- 
fication  prepares  bacteria  for  phagocytosis  we  know  nothing  that  is 
definite.  If  we  accept  the  view  of  Michalis,  that  ameboid  cells  react 
to  stimuli,  which  affect  their  surface  locally,  by  a  local  saponification 
of  their  lipoid  membrane  (ectoplasm),  and  that  this  leads  to  local 
changes  of  surface  tension,  which  in  turn  are  followed  by  mechanical 
surface  distortions  which  we  designate  as  ameboid  movements,  then 
we  may  imagine  that  the  primary  effect  of  the  opsonins  and  tropins 
upon  bacteria  may  be  such  that  the  bacterial  surface  is  so  influenced 
chemically  (sc.,  chemically-physically)  that  its  contact  with  the  lipoid 
ectoplasm  produces  the  same  effect  which  normally  emanates  from 
the  body  of  the  leukocyte  itself.  But  this  after  all  tells  us  very 
little  that  is  tangible.  So  much,  however,  is  certain,  that  opsonifi- 
cation  in  itself  does  not  impair  the  vitality  of  the  bacteria. 

While  the  discovery  of  the  opsonins  and  tropins  has  materially 
aided  our  conception  of  the  general  mode  of  action  of  the  leukocytes, 
and  has  demonstrated  the  relative  dependence  of  the  latter  upon  the 
presence  of  the  former  insofar  as  the  actual  process  of  phagocytosis 
is  concerned,  we  are  still  in  comparative  ignorance  of  the  mechanism 
by  which  leukocytes  are  attracted  toward  certain  bacteria  and  other 
organisms.  That  this  actually  occurs  is  a  matter  of  daily  observa- 
tion, every  abscess  formation  being  a  demonstration  of  the  event; 
for  pus  corpuscles  are  nothing  else  than  polynuclear  neutrophilic 
leukocytes  which  have  emigrated  from  the  bloodvessels  to  the  seat  of 


CHEMOTAXIS  65 

infection.  In  the  laboratory  the  same  can  be  shown  by  introducing 
tiny  capillary  tubes  filled  with  bacterial  cultures  (staphylococcus, 
typhoid  bacillus,  anthrax  bacillus,  etc.)  into  the  peritoneal  cavity 
of  frogs  and  leaving  them  for  twenty-four  hours.  At  the  expiration 
of  this  time  the  tubes  are  removed  and  examined  under  the  micro- 
scope, when  it  will  be  seen  that  the  ends  of  the  tubes  especially  are 
filled  with  leukocytes,  the  majority  of  which  contain  bacteria.  Every 
worker  in  the  clinical  laboratory  also  is  no  doubt  familiar  with  the 
precision  with  which  neutrophilic  leukocytes  will  enter  the  field  of 
vision  and  sooner  or  later  proceed  to  devour  an  extracellular  malarial 
organism  which  has  been  left  in  situ. 

Chemotaxis. — This  property  on  the  part  of  the  poly  nuclear  neutro- 
philic leukocytes  to  migrate  to  a  given  point  at  which  bacteria  or 
other  organisms  have  entered  the  body  is  generally  referred  to  chemo- 
tactic  influences  wThich  the  latter  exert  upon  the  leukocytes,  the  term 
chemotaxis  being  used  to  designate  a  certain  sensibility  on  the  part 
of  living  protoplasm  in  general  to  various  chemical  bodies;  it  is  a 
characteristic,  no  doubt,  which  the  leukocyte  has  inherited  from  its 
protozoan  ancestors. 

According  to  the  type  of  chemotaxis,  i.  e.,  the  existence  of  attract- 
ing or  repelling  influences  which  chemical  substances  exercise  upon 
living  cells,  we  speak  of  positive  and  negative  chemotaxis.  That  the 
latter  also  may  occur  in  bacterial  infections  is  an  established  fact, 
and  it  is  noteworthy  that  a  negative  effect  may  be  caused  by  a  viru- 
lent strain  of  the  same  organism  which  in  a  non-virulent  condition 
would  produce  positive  chemotaxis.  The  important  bearing  which 
the  type  of  chemotaxis  must  have  upon  the  production  of  an  infec- 
tion is,  of  course,  self-evident.  If  in  a  given  case,  in  which  the  main 
defence  lies  in  phagocytosis,  phagocytosis  cannot  occur  in  conse- 
quence of  negatively  chemotactic  influences,  it  is  clear  that  a  gen- 
eralized infection  must  be  the  outcome. 

Of  the  nature  of  the  substances  which  determine  the  chemotactic 
effect  we  know  relatively  little.  Living  bacterial  cells  are  mani- 
festly not  necessary  to  this  end,  for  we  obtain  the  same  collections 
of  leukocytes  in  the  peritoneal  cavity  of  frogs  (see  above)  with  dead 
organisms  and  even  with  the  soluble  products  of  bacterial  bodies,  as 
with  the  living  organisms  themselves,  and  it  has  long  been  known 
that  the  injection  of  sterilized  cultures  of  various  organisms  will 
lead  to  the  formation  of  sterile  abscesses  (Friedlander's  bacillus, 
5 


66        THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

staphylococci,  bacillus  subtilis,  bacillus  coli  communis,  anthracis, 
prodigiosus,  proteus  vulgaris,  etc.). 

Buchner,  to  whom  we  owe  so  much  of  our  information  on  this  sub- 
ject, was  the  first  to  suggest  that  the  chemotactic  influences  which 
are  here  manifestly  at  work  are  referable  to  bacterial  proteins,  and  he 
emphasized  that  abscess  formation  is  the  outcome  not  so  much  of 
the  presence  of  living  organisms,  but  of  their  dead  bodies  and  the  con- 
tained proteins.  According  to  Buchner,  moreover,  the  proteins  of  all 
bacteria  are  positively  chemotactic,  no  matter  whether  the  organisms 
in  question  are  otherwise  pathogenic  or  not. 

Of  the  mechanism  by  which  negative  chemotaxis  is  brought  to 
bear  upon  leukocytes  we  know  even  less  than  of  the  production  of 
pbsitive  chemotaxis.  That  the  virulence  of  the  organism  plays  an 
important  role  in  this  connection  is,  as  I  have  already  indicated, 
undoubted.  We  could  imagine  that  those  organisms  which  have 
developed  a  certain  degree  of  passive  resistance  in  consequence  of 
capsule  formation  are  less  liable  to  opsonification,  and  that  in  con- 
sequence phagocytosis  either  does  not  occur  at  all  or  does  so  only 
to  a  limited  extent.  In  such  a  case,  however,  we  could  hardly  speak 
of  negative  chemotaxis. 

A  beautiful  example  of  its  actual  occurrence,  however,  is  afforded 
if  the  attempt  is  made  to  increase  the  aggressivity  of  certain  organ- 
isms (in  the  sense  of  Bail)  by  passage  through  the  peritoneal  cavity 
of  a  series  of  animals,  and  transferring  with  the  bacteria  some  of  the 
peritoneal  exudate.  It  will  then  be  noted  that  whereas  the  exudate 
in  the  first  animal  is  rich  in  leukocytes  and  poor  in  bacteria,  most 
of  which  are  found  within  the  cells,  and  whereas  a  systemic  invasion 
has  not  taken  place,  the  latter  is  demonstrable  at  the  end  of  the 
series  and  simultaneously  we  find  the  peritoneal  cavity  filled  with 
a  thin  serous  fluid  which  is  swarming  with  bacteria,  but  is  almost 
free  from  leukocytes.  Evidently  there  were  strongly  positive  chemo- 
tactic influences  at  work  in  the  beginning  of  the  series,  while  at 
the  end  negative  chemotaxis  controls  the  situation. 

The  problem  then  seems  to  resolve  itself  into  a  question  of  differ- 
ence between  the  material  injected  into  the  first  as  compared  with  the 
last  animal  of  the  series.  The  bacteria  per  se  can  here  be  left  out  of 
sight,  as  the  same  result  is  obtained  if  for  each  injection  the  original 
culture  is  employed.  The  only  point  of  difference  then  is  the  absence 
of  aggressive  exudate  in  the  first  animal  of  the  series  and  its  presence 


VARIATION  IN  OPSONIC  CONTENT  OF  BLOOD  67 

in  the  subsequent  animals,  and  as  we  have  come  to  look  upon  Bail's 
aggressins  as  being  nothing  more  than  endotoxins  which  have  been 
set  free  after  the  death  of  the  organisms,  we  are  forced  to  the  con- 
clusion that  the  negatively  chemotactic  effect  must  be  referable  to 
such  substances.  In  this  sense  a  certain  parallel  undoubtedly  exists 
between  the  virulence  of  an  organism  and  the  chemotactic  effect 
which  it  produces. 

Phagocytosis  as  a  Defensive  Factor. — From  the  foregoing  consider- 
ations it  is  clear  that  the  leukocytes  can  rank  as  defensive  factors 
only  in  the  presence  of  opsonins  or  tropins,  and  providing  that  the 
aggressivity  of  the  invading  organisms  is  not  above  a  certain  level; 
it  accordingly  follows  that  any  factor  which  tends  to  lower  the  normal 
content  of  opsonins  or  prevents  the  prompt  formation  of  tropins  will 
virtually  be  equivalent  to  an  aggressive  influence  and  simulate  an 
increased  virulence  on  the  part  of  the  infecting  organisms.  The 
recognition  of  this  possibility  offers  an  explanation  of  the  formerly 
more  or  less  obscure  modus  operandi  of  some  of  those  factors  which 
the  clinician  speaks  of  as  predisposing  causes  of  disease. 

Everyone  is  familiar  with  the  serious  course  which  pneumonia 
is  apt  to  take  in  drunkards  and  with  the  liability  to  staphylococcus 
infections  of  diabetics.  Both  are  types  of  infection  in  which  phagocy- 
tosis plays  a  prominent  defensive  role,  and  we  have  already  sufficient 
evidence  to  show  that  both  alcoholism  and  diabetes  tend  to  lower 
the  opsonic  content  of  the  blood.  In  such  cases  we  could  readily 
understand  that  an  increased  virulence  on  the  part  of  the  infecting 
organism,  would,  in  itself,  not  be  necessary  to  produce  the  infection 
or  to  favor  its  generalization.  This,  indeed,  seems  to  be  Wright's 
attitude  in  reference  to  those  infections  in  general,  in  which  phago- 
cytosis is  the  mainstay  of  defence  on  the  part  of  the  body,  for  he 
expressed  the  belief  that  primary  infection  occurs  in  consequence 
of  a  lowered  opsonic  content  of  the  blood,  in  contradistinction  to 
the  idea  that  the  opsonic  content  drops  because  of  the  infection. 

On  the  other  hand,  we  can  now  understand  why  hyperemia  at  the 
point  of  infection  should  be  of  use  in  combating  the  infection,  no  mat- 
ter whether  the  hyperemia  be  the  direct  outcome  of  the  bacterial 
invasion  or  produced  artificially  (Bier's  method;  counter-irritation 
by  cautery  sinapisms,  applications  of  iodin,  turpentine,  etc.). 

Variation  in  Opsonic  Content  of  Blood. — That  the  opsonic  content  of 
the  blood  does  not  remain  constant  after  infection  has  once  begun 


68        THE  DEFENSIVE  FORCES  OF  THE  MACROORGANISM 

seems  to  stand  to  reason.  We  may  imagine  that  in  infections  of 
sufficient  magnitude  the  normal  opsonins  are  immediately  used  up 
to  a  greater  or  less  extent,  and  that  their  new  formation  as  well  as 
the  production  of  specific  tropins  then  begins.  We  know  but  little, 
however,  of  the  mechanism  by  which  this  is  brought  about,  and  by 
which  the  quantity  to  be  produced  is  regulated,  not  to  speak  of  the 
origin  of  the  bodies  in  question.  As  far  as  the  latter  point  is  con- 
cerned, some  observations  which  I  made,  together  with  Lamar,  led 
us  to  look  upon  the  leukocytes  themselves  as  the  possible  source 
of  the  opsonins,  but  the  evidence  was  not  conclusive.  On  the  other 
hand  there  can  be  but  little  doubt  that  the  production  of  opsonins 
and  tropins  is  caused  in  consequence  of  the  absorption  of  bacterial 
products.  This  phase  of  the  subject  has  been  thoroughly  investi- 
gated by  Wright  and  his  pupils,  who  found  that  the  injection  of 
killed  cultures  (vaccines)  of  various  bacteria  will  increase  the  opsonic 
content,  if  employed  in  suitable  amount,  whereas  overdoses  will 
cause  a  diminution. 

This  observation  throws  light  upon  the  remarkable  fluctuations 
in  the  opsonic  content  of  the  blood  that  have  been  noted  by  many 
observers  during  the  course  of  various  bacterial  diseases.  We  may 
well  imagine  that  these  fluctuations  are  brought  about  by  irregular 
absorption  of  bacterial  products,  and  it  naturally  suggests  itself  that 
in  those  diseases  particularly  which  are  characterized  by  a  certain 
chronicity,  and  in  which  the  opsonic  content  tends  to  be  low  (tuber- 
culosis, acne,  furunculosis,  gonorrheal  arthritis,  etc.),  it  might  be 
possible  to  raise  the  latter  by  artificial  means  (vaccination)  and  thus 
to  influence  the  infection  in  a  favorable  way.  This  assumption,  how- 
ever, presupposes  that  an  increased  content  of  normal  opsonins  or 
the  appearance  of  specific  tropins  would  be  the  only  factor  lacking 
to  insure  adequate  phagocytosis  and  thus  an  eradication  of  the  infect- 
ing organisms;  but,  as  I  have  already  mentioned,  there  is  evidence 
to  show  that  with  virulent  bacteria  at  least,  in  which  the  virulence 
is  due  to  capsule  formation,  phagocytosis  may  not  take  place  even 
though  opsonins  or  tropins  be  present  in  abundance,  and  it  is  indeed 
possible  that  this  factor  may  be  responsible  for  some  of  the  unsatis- 
factory results  which  vaccination  has  thus  far  yielded  in  the  curative 
treatment  of  bacterial  diseases. 


CHAPTER  VI 
THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

WE  have  seen  in  the  foregoing  chapter  that  the  outcome  of  a 
bacterial  invasion  will  of  necessity  be  influenced  by  the  phagocytic 
defence  of  the  body,  but  that  this  in  turn  is  largely  dependent  upon 
the  presence  of  certain  auxiliary  factors  in  the  body  fluids.  The 
recognition  of  the  interdependence  of  these  two  elements  is  most 
important,  as  it  has  in  a  measure  served  to  unite  the  two  opposing 
factions  of  immunity  students,  between  which  a  deadlock  had  prac- 
tically developed,  viz.,  the  cellular  school,  headed  by  Metschnikoff, 
and  the  humoral  school  of  Pfeiffer  and  Ehrlich,  who  looked  upon 
the  phagocytic  activity  of  the  leukocytes,  and  certain  bactericidal 
properties  of  the  body  fluids  respectively,  as  the  essential  protective 
mechanism  of  the  body  against  bacterial  infection. 

Alexins. — That  the  blood  serum  per  se  really  possesses  active  bac- 
tericidal properties  had  been  demonstrated  already  by  Fodor,  Nuttal, 
and  Buchner.  The  latter  ascribed  the  bactericidal  action  of  blood 
serum  to  substances  which  he  assumed  to  be  of  the  nature  of  ferments, 
and  which  he  designated  as  alexins.  Subsequent  studies,  which  are 
intimately  associated  with  the  names  of  Ehrlich  and  Morgenroth, 
Bordet,  Neisser,  and  Wechsberg,  etc.,  have  then  shown  that  the 
bactericidal  action  of  the  serum  is  dependent  upon  the  presence  of 
two  substances,  one  of  which  serves  as  a  connecting  link  between  the 
bacteria  and  the  second  substance,  and  which  has  been  variously 
termed  intermediary  body  (Ehrlich  and  Morgenroth),  substance 
sensibilisatrice  (Bordet),  fixateur  (Metschnikoff),  but  which  is  now 
generally  spoken  of  as  amboceptor  (Ehrlich),  whereas  the  second 
substance  has  been  designated  as  alexin  (Buchner  and  Bordet), 
cytase  (Metschnikoff),  or  complement  (Ehrlich  and  Morgenroth). 

Of  these  two  substances  the  complement  is  thermolabile  and 
destroyed  by  heating  for  30  minutes  at  56°  C.,  while  the  amboceptor 
is  relatively  thermostabile,  being  rendered  inactive  only  at  a  tempera- 
ture of  68°  to  70°  C.  The  complement  itself  is  incapable  of  combining 


70          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

with  the  bacteria,  whereas  the  amboceptor  is  readily  anchored  to  the 
organisms.  This  can  be  shown  by  treating  serum  with  killed  bac- 
teria (of  suitable  kind)  at  a  temperature  of  0°  C.,  and  subsequently 
removing  these  by  centrifugation.  The  absorption  of  the  ambo- 
ceptor is  then  shown  by  the  fact  that  such  serum  is  no  longer  capable 
of  causing  the  destruction  of  living  organisms  of  the  same  order, 
while  the  addition  of  such  extracted  but  fresh  serum  to  inactive 
(heated),  non-extracted  serum  will  render  this  actively  bactericidal. 
The  rationale  of  this  will  be  readily  understood  by  reference  to 
Fig.  1  and  bearing  in  mind  the  relative  thermostability  of  the  ambo- 
ceptor as  compared  with  the  complement. 

FIG.  1 


Bacterium  Amboceptor  Complement 

Mechanism  of  Interaction  between  Bacteria,  Amboceptor,  and  Com- 
plement.— Much  of  our  knowledge  of  the  mechanism  which  is  in- 
volved in  the  interaction  between  bacteria,  amboceptor,  and  com- 
plement has  been  obtained  from  a  study  of  the  closely  corresponding 
globulicidal  (hemolytic)  properties  which  certain  sera  possess  for 
red  corpuscles  of  animals  of  alien  species.  Working  with  washed 
corpuscles,  the  compound  character  of  the  hemolysin,  and  the  absorp- 
tion of  the  amboceptor  by  the  cells,  can  be  very  well  demonstrated 
as  follows:  Red  corpuscles  from  an  animal  of  a  suitable  species  are 
washed  free  from  serum  with  saline  (by  centrifugation),  and  then 
suspended  for  a  couple  of  hours  in  an  actively  hemolytic  serum,  the 
mixture  being  kept  at  a  temperature  of  from  0°  to  3°  C.  They  are 
then  thrown  down  again  by  means  of  the  centrifuge,  when  the  super- 
natant fluid  is  tested  at  body  temperature,  on  the  one  hand  against 
untreated  washed  corpuscles,  and  on  the  other  against  those  used 
in  the  extraction.  In  the  latter  case  hemolysis  will  result  because  the 
corpuscles  have  absorbed  the  hemolytic  amboceptor  and  are  now 
subjected  to  the  action  of  the  complement,  union  with  which  evi- 
dently does  not  occur  at  the  low  temperature  at  which  the  extrac- 
tion was  carried  out.  In  the  case  of  the  untreated  corpuscles  no 
hemolysis  is  observed,  because  the  amboceptor  has  been  previously 
removed  by  the  corpuscles  used  in  the  extraction,  showing  also  that 


PFEIFFER'S  EXPERIMENT  71 

complement  alone  possesses  no  hemolytic  properties.  That  ambo- 
ceptor  by  itself  is  similarly  inactive  is  proved  by  the  fact  that  the 
treated  corpuscles  per  se  will  not  be  hemolyzed  when  these  are 
suspended  in  saline. 

Demonstration  of  Bactericidal  Substances  in  Serum. — The  mere 
demonstration  of  the  presence  of  bactericidal  substances  in  a  given 
serum  is  a  relatively  simple  matter,  while  the  study  of  the  actual 
extent  of  its  destructive  action  meets  with  difficulties,  which  are 
largely  owing  to  the  fact  that  blood  serum,  while  it  may  be  bacteri- 
cidal, is  at  the  same  time  a  very  favorable  culture  medium,  and  that 
within  certain  limits  bacterial  destruction  and  bacterial  reproduction 
go  hand  in  hand.  The  values  which  are  thus  obtained  are  hence  of 
necessity  relative  values  only,  and  in  reality  merely  express  to  what 
extent  cell  destruction  predominates  over  cell  formation. 

Whether  or  not  a  given  serum  contains  bactericidal  substances 
can  be  determined  either  by  direct  microscopic  examination,  i.  e.,  by 
inoculating  tiny  little  tubes  of  fresh  serum  with  very  small  amounts 
of  a  certain  organism  (cholera  vibrio,  best)  and  observing  the  result- 
ant suspensions  in  the  hanging  drop  after  a  brief  incubation  at  37°  C., 
or  by  intraperitoneally  inoculating  a  guinea-pig  weighing  about  200 
grams  with  a  very  small  quantity  of  an  agar  culture  (less  than  yV 
milligram  =  -^V  oese — in  suitable  dilution — in  the  case  of  a  virulent 
culture  of  the  cholera  vibrio),  when  specimens  of  the  peritoneal  fluid 
are  removed  by  means  of  glass  capillaries  at  intervals  of  10,  20,  or 
30  minutes,  etc.,  and  examined  either  directly  in  the  hanging  drop 
or  after  staining,  in  the  dried  smear.  With  either  method  it  will  be 
observed  that  the  organisms  at  first  lose  their  motility  and  then 
contract  to  little  granules  which  in  the  beginning  are  highly  refrac- 
tive, but  gradually  become  paler  and  paler,  until  they  dissolve  alto- 
gether. At  first  the  granules  can  still  be  stained  with  anilin  dyes, 
but  as  the  process  of  destruction  proceeds  they  become  paler  and 
paler,  until  at  last  they  are  no  longer  demonstrable.  As  Wassermann 
very  appropriately  remarks,  the  granules  melt  away  like  wax  in 
boiling  water. 

Pfeiffer's  Experiment. — Still  more  striking  results  will  be  obtained 
if  the  animal  is  simultaneously  injected  with  a  small  quantity  (about 
1  milligram)  of  serum  from  another  animal  which  has  been  previously 
rendered  highly  immune  against  the  organism  in  question  by  vaccina- 
tion (which  see).  In  such  a  case  a  much  larger  quantity  of  bacteria 


72          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

may  be  injected  (1  oese)  with  impunity,  and  it  will  be  observed 
that  notwithstanding  the  large  dose  no  bacteria  will  be  found  in  the 
peritoneal  cavity  at  the  expiration  of  one  hour  (Pfeiff er's  experiment) . 
The  same  result  will  be  obtained  if  instead  of  using  a  normal  guinea- 
pig  and  injecting  some  immune  serum  together  with  the  bacteria, 
these  are  introduced  by  themselves  into  a  previously  immunized 
animal. 

The  reason  why  bacteriolysis  will  be  so  much  more  extensive 
in  the  presence  of  immune  serum  is  the  fact  that  as  a  result  of 
infection  (vaccination,  immunization)  the  amboceptor  content  of 
the  blood  serum  is  materially  increased.  The  complement  which  is 
necessary  for  the  experiment  is  normally  present  in  the  living  animal. 
In  its  absence,  of  course,  bacteriolysis  could  not  take  place,  and 
as  complement  readily  becomes  inactive  outside  of  the  body,  after 
standing  even  for  a  relatively  short  time  at  body  or  room  tempera- 
ture, it  is  essential  if  the  experiment  is  conducted  in  vitro  that  only 
perfectly  fresh  serum  be  used.  Otherwise  bacteriolysis  will  not  occur, 
even  though  the  serum  may  be  rich  in  natural  amboceptors. 

Quantitative  Estimation  of  Bactericidal  Substances. — The  quanti- 
tative estimation  of  the  content  in  bactericidal  substances  of  a  given 
serum  is  most  conveniently  carried  out  by  starting  with  a  suspen- 
sion, of  known  number,  of  the  organism  to  be  examined,  and  inocu- 
lating tubes  containing  known  amounts  of  serum,  after  which  these 
are  incubated  for  a  certain  period  of  time  and  plates  are  prepared 
in  which  the  number  of  surviving  organisms  is  finally  determined 
by  a  direct  count.  As  serum  is  in  itself  an  admirable  culture  medium 
for  most  organisms  it  is,  of  course,  essential  to  reduce  this  factor 
as  much  as  possible  in  the  experiment.  To  this  end  one  can  either 
determine  the  total  number  of  bacteria  which  is  completely  killed 
by  a  given  amount  of  serum  in  a  given  length  of  time,  or  one  can 
determine  the  extreme  degree  of  dilution  in  which  a  given  serum  will 
still  exercise  a  bactericidal  effect,  or  one  may  determine  the  maximal 
bactericidal  effect,  which  is  observed  after  different  intervals  of 
time.  The  general  arrangement  of  such  a  test  is  apparent  from 
the  following  example,  which  is  taken  from  Wright,  and  which 
represents  the  titration  of  a  given  serum  against  cholera  vibrios 
on  the  one  hand  and  typhoid  bacilli  on  the  other: 

A.  Determination  of  the  Strength  of  the  Bacterial  Emulsion  which 
Serves  as  a  Basis  of  the  Experiment. — The  emulsion  in  question  is 


ESTIMATION  OF  BACTERICIDAL  SUBSTANCES  73 

diluted  in  the  proportion  of  1  to  100,000  and  5  c.c.  of  the  resultant 
dilution  distributed  over  the  surface  of  plates.  The  number  of 
developing  colonies  is  then  counted.  The  result  shows  the  following: 


5  c.c.  of  a  1  to  100,000        1  c.c.  of  the  undiluted  emulsion 

Type.                                                      dilution  contain:  hence  contains: 

Cholera  vibrios    ....     Plate  I  20.0  organisms 

Plate  II  21.0  organisms 

Average  21.5  organisms         410,000  organisms 

Typhoid  bacilli    ....     Plate  I  21.0  organisms 

Plate  II  30.0  organisms 

Average  25.5  organisms         510,000  organisms 


B.  Estimation  of  the  Bactericidal  Strength  of  the  Serum. — Equal 
quantities  (1  c.c.)  of  the  undiluted  serum  and  of  various  dilutions 
of  the  original  emulsion  are  mixed  and  after  twenty-four  hours' 
exposure  plated  out,  when  the  number  of  developing  colonies  is 
counted. 

Dilution  of  the       Number  of 
bacterial  developing 

Type.  emulsion.  colonies.  1  c.c.  of  serum  thus  kills: 

Cholera undiluted  1 

1  to  10  0 

1  to  100  0          About  410,000  organisms 

1  to  1000  0 

1  to  10000  0 

1  to  100000  0 

Typhoid 1  to  10  8 

1  to  100  1 

1  to  1000  0          About  5100  organisms 

1  to  10000  0 

1  to  100000  0 


A  good  idea  of  the  progress  of  bacteriolysis,  and  the  gradual  gain 
of  reproduction  over  destruction,  when  the  amount  of  serum  was 
insufficient  at  the  start  to  kill  all  the  organisms,  as  also  of  the 
differing  resistance  which  different  strains  of  organisms  offer  to  the 
destructive  forces  of  the  serum,  may  be  had  from  the  study  of  the 
following  table  (taken  from  Trommsdorff).  The  figures  in  general 
represent  the  variations  noted  in  different  tests;  those  of  the  a 
series  were  obtained  with  a  typhoid  strain  that  had  been  freshly 


74          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

cultivated  from  a  patient,  while  the  b  series  has  reference  to  a 
common  laboratory  strain. 

After  2  to  3  After  5  to  6  After  24  to  25 

Original  count.  hours.  hours.  hours. 

Series  a     ...     10221  to  12256    2074  to  4275    3217  to  4664    8344  to  39125 
Series  6     ...       2016  to  6586  0  to  0  0  to  0  0  to  0 

Specificity  of  Normal  Antibacterial  Amboceptors  and  Complement. 
—Practically  important  is  the  fact  that  the  normal  thermostabile 
antibacterial  amboceptors  are  specific,  as  can  be  shown  by  treating 
an  inactivated  serum  with  cholera  vibrios,  for  example,  when  it  will 
be  noted,  after  removal  of  the  organisms  by  centrifugation,  that 
the  fluid,  upon  the  addition  of  suitable  complement,  has  lost  the 
power  of  causing  the  destruction  of  newly  added  cholera  organisms, 
while  it  is  still  destructive  for  typhoid  organisms.  In  other  words,  the 
anticholera  amboceptors  have  been  extracted,  while  the  antityphoid 
amboceptors  have  not  been  affected  by  the  first  extraction. 

Detailed  analytical  studies  of  the  different  kinds  of  amboceptors 
contained  in  normal  human  serum  are  apparently  lacking,  but  it 
appears  from  what  has  been  done  that  whereas  anticholera,  anti- 
typhoid, anticolon,  and  antidysentery  amboceptors  are  usually  to  be 
found,  normal  blood  possesses  no  bactericidal  power  over  the  various 
staphylococci,  the  pneumococcus,  micrococcus  melitensis,  bacterium 
pestis,  bacillus  xerosis,  and  the  diphtheria  bacillus,  nor  do  such 
substances  appear  in  the  human  being  as  the  result  of  infection. 

Providing  that  suitable  complement  is  present,  bacteriolysis 
will,  of  course,  occur  whenever  blood  serum  containing  a  given 
amboceptor  is  brought  in  contact  with  the  corresponding  bac- 
teria. As  a  general  rule  the  complement  of  the  same  serum  as  that 
containing  the  amboceptor,  or  at  any  rate  that  from  an  animal 
of  the  same  species,  will  be  found  to  be'  effective,  but  there  are  a 
number  of  curious  exceptions.  The  serum  of  the  human  being,  of  the 
ox,  and  of  the  dog  thus  contains  anti-anthrax  amboceptors,  which  are 
not  activated  by  the  corresponding  complement.  Such  sera  per  se 
have  no  bactericidal  action  whatever  for  the  organism  in  question, 
while  the  addition  of  a  little  rabbit  serum  renders  them  active.  The 
recognition  of  the  fact  that  the  serum  of  an  animal  of  a  different 
species  may  contain  complement  that  will  activate  a  given  ambo- 
ceptor is  of  great  practical  interest,  as  it  is  often  technically  more 
convenient  to  use  as  complement  the  blood  of  a  certain  animal  rather 


ANTIBACTERIAL  AMBOCEPTORS  AND  COMPLEMENT      75 

than  that  of  another;  but  it  is  essential  to  remember  that  considerable 
differences  in  the  behavior  of  such  sera  exist,  and  that  the  sera  from 
certain  animals  only  will  serve  to  activate  others. 

These  interrelations  (worked  out  for  hemolytic  amboceptors  on 
the  one  hand  and  bacteriolytic  amboceptors  on  the  other,  both 
normal  and  immune)  are  shown  in  the  following  table: 


Type  of  cell. 

Red  cells  of  rabbit  . 
Red  cells  of  rabbit  .      . 

Red  cells  of  guinea-pig 


Red  cells  of  sheep  . 
Red  cells  of  chicken 
Dysentery  bacillus 
Typhoid  bacillus  . 
Proteus  .... 
Proteus  .... 

Anthrax'  bacillus 

Anthrax  bacillus 
Anthrax  bacillus 
Anthrax  bacillus 
Anthrax  bacillus  . 

Vibrio  Metschnikoffi 
Vibrio  Metschnikoffi 

Vibrio  Metschnikoffi 
Vibrio  cholerse  . 
Dysentery  bacillus 
Typhoid  bacillus 
Typhoid  bacillus 
Typhoid  bacillus     . 
Typhoid  bacillus     . 
Typhoid  bacillus     . 


Amboceptor-containing 
serum  of  the 


Dog  (normal) 


Corresponding  complement 
found  in  the 


I  Guinea-pig 
Ox 
Goat 
Sheep 


C  Guinea-pig 

Ox 

(normal) 

]  Rabbit 

iRat 

Guinea-pig 

Man 

Ox 

(normal) 

Rat 

Horse 

Sheep  (less  markedly) 

Rabbit 

(immune) 

Guinea-pig 

Rabbit 

(immune) 

Guinea-pig 

Goat 

(normal) 

Horse 

Rabbit 

(normal) 

Guinea-pig 

Dog 

(normal) 

Rabbit 

Cat 

(normal) 

Rabbit 

Dog 

(normal)            /Rabbit 

IRat 

Man 

(normal) 

Ox 

(normal) 

~D~± 

Pig 
Goat 

(normal)             |  i^" 
Horse 
(normal) 

Chicken 

(immune) 

Pigeon 

[Pigeon 

Goose 

(immune) 

Rabbit 

Goat 

Goat 

(immune) 

Rabbit 

Goat 

(immune) 

Rabbit 

Horse 

(immune) 

Man 

Man 

(immune) 

Rabbit 

Donkey 

(immune) 

Rabbit 

Horse 

(immune) 

Rabbit 

Rabbit 

(immune) 

Goat 

Dog 

(immune) 

Guinea-pig 

76          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

Origin  of  Bacteriolytic  Amboceptors. — Regarding  the  origin  of  the 
bacteriolytic  amboceptors,  our  knowledge  is  as  yet  very  meager. 
The  researches  of  Pfeiffer  and  some  of  his  pupils  suggest  that  the 
spleen  is  possibly  more  actively  concerned  in  their  production  than 
any  other  organ,  but  there  is  also  evidence  to  show  that  they  may 
be  formed  in  the  tissues  at  large. 

Relative  Importance  of  Amboceptor  and  Complement. — As  to  the 
relative  importance  of  amboceptor  and  complement,  opinions  differ. 
Ehrlich  regards  the  amboceptor  merely  as  an  indifferent  connecting 
link  between  the  bacteria  and  the  complement,  and  Bordet  also 
views  the  complement  as  the  essential  factor  in  bacteriolysis,  the 
amboceptor  playing  the  role  of  a  mordant  or  activator;  on  the  other 
hand,  Pfeiffer  emphasizes  the  greater  importance  of  the  amboceptor, 
and  likens  its  role  to  that  of  a  preferment  with  the  complement 
playing  the  role  of  the  corresponding  kinase.  In  support  of  this 
view  he  calls  attention  to  the  fact  that  as  a  result  of  immunization 
(infection,  vaccination)  only  the  amboceptor  is  increased,  while  the 
complement  content  is  not  affected,  and  further,  also,  that  during 
the  process  of  hemolysis  (which  is  in  every  respect  closely  related 
and  directly  comparable  to  bacteriolysis)  the  complement  is  active, 
not  in  proportion  to  its  absolute  amount,  but  in  accordance  with 
its  concentration;  this  would  be  quite  in  harmony  with  the  supposi- 
tion that  its  action  in  reference  to  the  amboceptor  is  essentially  that 
of  a  catalyzing  agent. 

Origin  and  Structure  of  Complement. — Regarding  the  origin  and 
structure  of  the  complement  our  knowledge  is  likewise  imperfect, 
though  somewhat  more  definite  than  that  concerning  the  ambo- 
ceptor. While  originally  it  was  viewed  as  a  single  substance,  Ferrata 
has  shown  that  on  dialysis  the  complement  separates  into  two  com- 
ponents, one  of  which  is  carried  down  in  the  precipitate  of  globulins — 
the  so-called  middle  piece  (Mittelstiick),  while  the  other  remains  in 
solution — the  end  piece  (Endstuck).  Of  the  two,  as  the  term  indi- 
cates, the  first  named  (Mittelstiick)  unites  with  the  combination  of 
bacteria  (sc.,  blood  corpuscles)  and  the  corresponding  amboceptor, 
while  the  end  piece  only  exercises  its  activity  after  this  union  has  been 
effected.  Either  fraction  alone  possesses  no  bactericidal  properties 
in  the  presence  of  a  suitable  amboceptor,  though  it  appears  that 
either  component  can  in  a  measure  supplement  the  action  of  the 
other,  in  the  sense  that  a  very  small  quantity  of  the  globulin  fraction 


CHEMICAL  NATURE  OF  AMBOCEPTOR  AND  COMPLEMENT      77 

of  the  serum  (middle  piece)  is  sufficient  to  effect  bacteriolysis,  pro- 
viding that  a  sufficiently  large  amount  of  the  end  piece  is  present; 
and  vice  versa,  the  germicidal  properties  of  the  end  piece  are 
enhanced  by  increasing  the  amount  of  the  middle  piece.  Since  the 
middle  piece  of  different  animal  sera  is  interchangeable,  the  thought 
suggests  itself  that  the  two  complement  components  in  the  living 
animal  are  actually  present  as  separate  substances.  The  apparent 
absence  of  complement  in  various  sera  which  we  have  noted  above, 
might  thus  be  due  to  the  absence  or  deficiency  in  the  end  piece 
only,  rather  than  to  actual  absence  of  complement  as  a  whole. 

Complementoid. — This  conception  of  the  duality  of  the  complement 
is  quite  in  accord  with  the  original  view  of  Ehrlich  and  Morgenroth 
regarding  its  structure,  according  to  which  the  substance  in  question 
contained  a  combining  (haptophoric)  group  which  effects  the  union 
with  the  amboceptor  and  a  second  (toxophoric  or  zymophoric)  group 
to  which  the  action  of  the  complement  is  essentially  due.  When 
this  latter  group  is  destroyed,  while  the  first  remains  active,  so-called 
complementoid  results,  which  would  mean  in  more  modern  parlance 
that  the  middle  piece  remains,  while  the  end  piece  has  been  destroyed 
or  altered  so  as  to  be  rendered  inactive. 

Chemical  Nature  of  Amboceptor  and  Complement. — Regarding  the 
chemical  nature  of  amboceptor  and  complement  our  knowledge  is 
very  meager.  Liebermann  has  pointed  out  that  a  certain  analogy 
exists  between  the  action  of  amboceptor  and  oleic  acid.  Oleic  acid 
and  hog  serum  together  will  thus  cause  the  almost  instantaneous 
hemolysis  of  hog  corpuscles,  while  the  serum  by  itself  is  inactive, 
and  the  same  quantity  of  oleic  acid  alone  brings  about  the  lysis  of 
the  corpuscles  only  very  slowly.  This  observation  is  suggestive, 
but  can  hardly  be  taken  to  prove  the  identity  of  the  hemolytic 
amboceptor  and  oleic  acid,  especially  in  view  of  the  highly  specific 
action  of  the  various  amboceptors. 

Complement,  on  the  other  hand,  has  been  viewed  as  a  compound 
of  albumin  with  a  lipoid  (Noguchi  and  Liebermann).  Noguchi  thus 
found  that  mixtures  of  soaps  and  inactivated  guinea-pig  serum,  while 
inactive  by  themselves,  caused  the  hemolysis  of  red  corpuscles  which 
had  been  previously  treated  with  amboceptor.  He  himself,  however, 
points  out  that  the  action  of  such  artificial  complement  is  materially 
slower  than  that  of  the  native  serum.  But  notwithstanding  this  and 
other  points  of  similarity  between  active  and  artificial  complement, 


78          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

such  as  the  spontaneous  disappearance  of  the  complementary  prop- 
erties on  standing,  inactivation  at  56°  C.,  absence  of  a  hemolytic 
effect  at  0°  C.,  etc.,  the  proof  that  complement  is  in  reality  a  lipoid- 
albumin  product  has  not  yet  been  furnished. 

Origin  of  Complement. — Regarding  the  origin  of  the  complement, 
Buchner  and  Metschnikoff  both  thought  that  it  was  derived  from 
the  leukocytes,  but  while  Buchner  looked  upon  the  substance  as  a 
secretory  product  of  the  living  cells,  Metschnikoff  claimed  that  com- 
plement is  only  formed  when  the  cell  dies,  during  the  process  of 
blood  coagulation;  and  that  it  does  not  exist  preformed  in  the  circu- 
lating blood.  An  enormous  amount  of  labor  has  been  expended  to 
support  this  view  of  Metschnikoff  on  the  one  hand  and  to  disprove 
it  on  the  other.  As  a  result  it  may  now  be  regarded  as  a  fairly  well 
established  fact  that  the  normal  body  fluids  contain  free  complement 
even  when  there  is  no  evidence  that  leukocytic  degeneration  has 
taken  place. 

The  long  discussed  question,  also,  whether  or  not  the  blood  plasma 
contains  free  complement,  may  now  be  answered  in  the  affirmative. 
On  the  other  hand,  there  can  be  no  doubt  that  bactericidal  substances 
can  be  extracted  directly  from  the  leukocytes.  This  can  be  shown 
in  the  following  manner :  An  aseptic  exudate  is  produced  in  animals 
by  the  intrapleural  injection  of  aleuronat,  when  the  cellular  elements, 
which  are  mostly  poly  nuclear  leukocytes  (Metschnikoff 's  micro- 
phages),  are  thoroughly  washed  with  saline,  repeatedly  frozen  and 
thawed  and  the  resultant  material  allowed  to  stand  at  body  tem- 
perature. After  a  while  it  can  then  be  shown  that  this  extract  is 
quite  rich  in  bactericidal  substances  and,  like  the  fresh  blood  serum, 
loses  its  action  on  exposure  to  higher  temperatures.  But  on  com- 
paring the  behavior  of  such  leukocytic  extracts  with  normal  b.acteri- 
cidal  sera  certain  points  of  difference  appear,  nevertheless,  which 
suggest  that  the  substances  which  are  operative  on  the  two  sides 
may  not  be  identical. 

Apart  from  the  different  temperature  at  which  inactivation  takes 
place  and  the  slower  action  of  the  leukocytic  extracts  which,  more- 
over, can  progress  in  the  absence  of  neutral  salts  (contrary  to  the 
bacteriolytic  sera),  it  is  especially  noteworthy  that  certain  organ- 
isms, such  as  the  cholera  vibrio  and  the  typhoid  bacillus,  which  are 
very  susceptible  to  the  action  of  bacteriolysins,  are  hardly  affected 
by  leukocytic  extracts.  The  latter,  moreover,  contain  no  substances 


LEU  KINS  79 

which  are  capable  of  activating  inactivated  anticholera  or  anti- 
typhoid sera,  i.  e.,  sera  containing  the  corresponding  bacteriolytic 
amboceptors,  but  deprived  of  their  active  complement. 

We  are  thus  forced  to  conclude  that  the  leukocytic  origin  of  com- 
plement has  not  been  proved,  but  we  are  also  forced  to  admit  that 
no  other  cells  have  as  yet  been  shown  to  form  complement.  As  long 
as  the  latter  was  regarded  as  a  single  substance  this  negative  search 
might  very  naturally  lead  one  to  think  that  our  technique  may  have 
been  imperfect,  but  now,  when  we  know  that  what  we  call  comple- 
ment is  very  evidently  composed  of  two  constituents,  which  can  be 
separated  from  one  another  and  then  reunited  to  reform  active  com- 
plement, the  possibility  suggests  itself  that  these  two  components 
may  have  a  separate  origin,  and  it  will  accordingly  be  necessary  to 
repeat  the  search  from  this  standpoint. 

Leukins. — Since  the  leukocytes  have  been  virtually  eliminated  as 
the  source  of  the  serum  complement,  in  the  older  sense  of  the  word, 
while  their  bactericidal  action  toward  certain  organisms  at  least  is 
an  established  fact,  we  are  forced  to  the  conclusion  that  the  body  has 
at  its  disposal  still  other  defensive  factors  than  those  with  which  we 
have  thus  far  become  acquainted.  To  what  extent  such  substances 
occur  in  the  tissues  at  large  still  remains  to  be  determined.  A  priori 
it  would  seem  reasonable  to  expect  that  they  might  be  present  in  all 
cells,  but  thus  far  their  production  by  the  leukocytes  only  has  been 
satisfactorily  established. 

These  leukocytic  alexins,  as  we  may  term  them,  using  the  word 
alexin  in  the  original  sense,  viz.,  synonymously  with  ''protective 
substances,"  have  been  variously  described  as  leukins  and  leukocytic 
endolysins  by  Schneider  and  Peterson  respectively,  and  the  former 
seems  to  have  proved  quite  conclusively  that  these  bactericidal  sub- 
stances are  actually  secreted  by  the  leukocytes,  as  Buchner  originally 
claimed  for  the  common  alexins  of  the  serum.  This  was  demon- 
strated by  placing  leukocytes  for  30  minutes  in  diluted  blood  serum 
(5  per  cent,  in  normal  salt  solution),  when  it  could  be  shown  that  the 
solution  had  developed  very  active  bactericidal  properties.  During 
this  process  the  leukocytes  were  not  destroyed,  but  could  be  made 
to  furnish  additional,  equally  active  extracts  without  impairment  of 
their  vital  functions  (power  of  phagocytosis,  locomobility ) .  If  the 
same  experiments  were  carried  on  in  an  atmosphere  of  carbon  dioxide 
the  solutions  developed  no  bactericidal  properties,  while  a  trans- 


80          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

f erence  of  the  leukocytes  to  ordinary  conditions  again  led  to  an  active 
production  of  "leukins,"  the  cells  having  apparently  not  been 
damaged  by  their  exposure  to  the  carbon  dioxide.  Corresponding 
results  were  obtained  with  Bier's  congestive  lymph,  and  satisfactory 
proof  thus  furnished  that  these  bodies  are  formed  in  vivo  as  well  as 
in  vitro.  Schneider,  hence,  naturally  concludes  that  the  beneficial 
effect  obtained  with  this  method  of  treatment  is  probably  due  to 
the  increased  production  of  these  substances. 

What  the  determining  factor  is  that  causes  the  secretion  of  leukins 
is  unknown,  but  we  may  well  imagine  that  a  special  stimulus  may  here 
be  operative,  and  that  such  substances  may  be  liberated  from  the 
tissues  at  large  under  various  conditions  which  need  not  necessarily 
be  pathological. 

While  the  above-mentioned  bactericidal  substances,  namely,  the 
bacteriolysins  of  the  serum  and  the  leukins  or  endolysins  of  the  leuko- 
cytes, unquestionably  exist  as  such  in  the  plasma,  another  substance 
or  group  of  substances  which  may  likewise  be  viewed  as  protective 
agents  or  alexins,  in  the  wider  sense  of  the  word,  are  formed  only 
during  the  process  of  coagulation  from  the  blood  platelets,  as  has 
been  satisfactorily  demonstrated  by  Gruber  and  Futaki.  Their 
action,  however,  seems  to  be  directed  almost  exclusively  against 
the  anthrax  bacillus  and  its  congeners. 

Summary. — To  sum  up  then:  we  have  become  acquainted  with 
various  defensive  agents  on  the  part  of  the  animal  body,  any  one  or  all 
of  which  may  become  operative  after  infection  has  once  occurred, 
i.  e.,  after  a  given  organism  has  penetrated  through  the  external 
epithelial  barriers  of  the  body;  and  knowing  some  of  the  offensive 
weapons  of  the  invaders,  we  can  form  a  conception  of  the  manner  in 
which  systemic  invasion  may  take  place  or  in  which  it  may  be  pre- 
vented. A  great  deal  will,  of  course,  depend  upon  the  quantitative 
relations  existing  between  the  offensive  and  defensive  factors  which 
are  engaged  in  the  strife. 

Offensive-defensive  Mechanism  in  Infections  with  Necroparasites.— 
If  the  invader  is  actually  open  to  attack  at  the  point  of  infection  by 
those  agents  which  are  at  the  disposal  of  the  host,  a  successful  resist- 
ance is  at  least  possible,  which  may  carry  with  it  the  recovery  of  the 
infected  individual.  This,  however,  is  not  necessarily  the  case.  For 
we  have  seen  already  that  some  organisms,  such  as  the  tetanus 
bacillus,  are  capable  of  producing  poisons  of  such  potency  that 


MECHANISM  IN  INFECTIONS  WITH  TRUE  PARASITES      81 

infinitesimally  small  quantities  are  sufficient  to  produce  death; 
whose  manner  of  action,  moreover,  is  such  that  the  focal  infection 
may  long  have  ceased  to  exist,  while  the  toxin  is  being  conveyed  to 
and  brought  into  contact  with  cells,  damage  to  which  leads  to  the 
death  of  the  patient. 

In  an  infection  of  this  sort  the  local  superiority  of  those  defensive 
forces  of  the  host  with  which  we  have  thus  far  become  acquainted, 
over  the  purely  vegetative  forces  of  the  invader,  is  evidently  totally 
insufficient  to  preserve  the  life  of  the  infected  individual,  unless, 
indeed,  the  disproportion  between  the  number  of  the  infecting  germs, 
and  the  protective  forces  at  the  point  of  attack  should  be  so  greatly 
in  favor  of  the  latter,  that  no  multiplication  of  the  bacteria  occurs  at 
all,  and  then  only  provided  that  the  number  of  invading  organisms 
is  so  small  that  the  amount  of  toxin  which  they  could  secrete  before 
being  killed  would  be  insufficient  to  cause  death.  Theoretically  this 
possibility  could  certainly  exist.  Whether  it  enters  into  considera- 
tion practically  is  beyond  our  knowledge. 

This  type  of  infection  illustrates  two  points  very  well,  viz.,  that  the 
destruction  of  the  invading  bacteria  at  the  point  of  entry  does  not 
necessarily  prevent  the  development  of  symptoms  of  systemic  dis- 
ease and  even  of  death,  and  that  the  protective  forces  with  which 
we  have  thus  far  become  acquainted  are  inadequate  to  counteract 
the  deleterious  influence  of  toxins  of  this  order. 

Offensive-defensive  Mechanism  in  Infections  with  True  Parasites.— 
In  infections  with  organisms  like  the  anthrax  bacillus  the  situation 
is  altogether  different.  The  picture  which  is  here  seen  has  been 
analyzed  with  great  care  by  Bail,  whose  account  I  am  here  following 
in  some  detail. 

If  a  guinea-pig  or,  still  better,  a  rabbit  is  injected  intraperitoneally 
with  a  moderate  amount  of  a  broth  culture  (J  to  1  c.c.)  of  the  anthrax 
bacillus,  and  small  specimens  of  the  peritoneal  fluid  are  removed  from 
time  to  time,  it  will  be  observed,  after  a  short  while,  that  the  bacilli 
show  external  marks  of  degeneration,  and  are  being  extensively 
taken  up  by  the  leukocytes,  which  have  appeared  in  large  numbers, 
and  undergo  intracellular  degeneration. 

Evidently  some  of  those  defensive  forces  with  which  we  have  just 

become  familiar  (opsonins,  alexins)  are  here  at  work,  and  unless 

the  number  of  organisms  injected  has  been  too  large,  these  normal 

protective  forces  are  apparently  sufficient  to  successfully  combat  the 

6 


82          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

infection,  for  it  will  be  observed  that  after  a  certain  length  of  time  the 
peritoneal  cavity  is  seemingly  free  from  bacteria  and  may  remain  so 
for  twenty-four  hours  or  longer.  Conditions  are,  however,  in  reality 
not  at  all  so  favorable  as  appearances  would  lead  one  to  think,  for 
presently  organisms  begin  to  reappear  and  to  multiply  rapidly  in 
spite  of  the  fact  that  leukocytes  are  present  in  abundance.  Phago- 
cytosis then  no  longer  occurs  and  signs  of  extracellular  degeneration 
are  altogether  wanting.  Simultaneously  bacilli  have  appeared  in  the 
circulating  blood  and  multiply  here  also  without  hindrance. 

It  might  be  argued  that  this  change  in  conditions  was  brought 
about  through  a  gradual  loss  of  those  defensive  substances  in  the 
peritoneal  fluid  of  the  guinea-pig,  to  which  the  primary  successful 
resistance  was  due;  but  this  is  disproved  by  the  fact  that  if  a  new  lot 
of  "culture"  bacilli,  like  those  used  to  bring  about  the  infection  in  the 
beginning,  be  now  injected,  these  will  be  destroyed  as  readily  and  in 
the  same  manner  as  the  first.  Evidently,  then,  the  same  defensive 
factors  of  the  host  are  still  available. 

The  conclusion,  hence,  suggests  itself  that  some  change  may  have 
taken  place  in  the  bacteria,  and  microscopic  examination  shows,  as 
a  matter  of  fact,  that  the  newly  developed  brood  really  differs  from 
the  stock  culture  in  having  become  encapsulated.  Further  experi- 
ments, however,  show  that  the  capsule  formation  in  itself  does  not 
explain  the  result,  for  if  some  of  these  capsulated  bacilli  are  injected 
into  the  peritoneal  cavity  of  another  guinea-pig,  they  do  not  imme- 
diately multiply  without  hindrance;  but,  as  in  the  beginning  of  the 
first  experiment,  they  undergo  extensive  extracellular  degeneration, 
and  here,  as  there,  the  peritoneal  cavity  may  be  temporarily  freed  of 
bacteria,  although  phagocytic  destruction  apparently  does  not  take 
place. 

This  shows  clearly  that  while  the  phagocytic  forces  are  no  longer 
available  in  combating  the  capsulated  organisms,  some  of  the  normal 
extracellularly  active  lytic  forces  are  still  available.  But,  if  so,  why 
do  they  remain  inactive  in  the  body  of  the  first  animal?  It  can 
easily  be  shown  that  guinea-pig  serum  in  itself  has  little  or  no  bacteri- 
cidal power,  so  far  as  the  anthrax  bacillus  is  concerned.  If,  then,  the 
peritoneal  exudate  has  this  to  a  marked  extent  the  thought  naturally 
suggests  itself  that  the  destruction  of  the  bacilli  may  be  the  out- 
come of  a  combined  serum-leukocyte  effect — possibly  in  the  sense 
of  Schneider's  leukins  or  Peterson's  leukocytic  endolysins. 


MECHANISM  IN  INFECTIONS  WITH  TRUE  PARASITES     83 

If  Schneider's  experiments  permit  the  inference  that  these  sub- 
stances are  formed  through  the  secretory  activity  of  the  living  cells, 
then  the  possibility  also  suggests  itself  that  this  activity  may  be 
impaired  or  paralyzed  as  a  consequence  of  bacterial  action,  and  this 
is  what  Bail  actually  claims  for  his  aggressins.  He  has  shown, 
moreover,  that  the  peritoneal  exudate  has  lost  its  anthracoccidal 
properties  even  before  animalized  bacteria  are  there  demonstrable, 
and  that  organisms  are  at  this  time  already  present  in  the  internal 
organs  and  notably  the  spleen.  He  accordingly  develops  the  follow^- 
ing  picture  of  what  actually  happens:  In  consequences  of  the  normal 
protective  forces,  i.  e.,  the  phagocytic  action  and  the  secretion  of 
leukins  on  the  part  of  the  attracted  leukocytes,  the  majority  of  the 
injected  organisms  are  killed  off  soon  after  their  introduction.  Some 
of  them  escape,  however,  and  in  the  internal  organs  and  notably 
the  spleen  these  find  a  relatively  safe  refuge.  Here  they  assume  their 
"animalized"  (infectious)  state  and  begin  the  secretion  of  aggressins. 
The  latter  are  distributed  through  the  general  circulation  and  also 
reach  the  peritoneal  cavity,  where  they  inhibit  the  secretory  activity 
of  the  leukocytes  and  then  furnish  suitable  conditions  for  the  multi- 
plication of  any  surviving  bacilli  that  may  yet  be  present.  The 
animal  is  then  stripped  of  its  entire  defensive  mechanism  and 
now  succumbs  to  the  generalized  infection. 

That  the  peritoneal  fluid  actually  contains  substances  which  can 
prevent  the  liberation  of  leukins,  at  the  time  of  the  second  invasion, 
Bail  has  demonstrated  beyond  a  doubt.  For,  on  adding  peritoneal 
exudate  from  an  infected  guinea-pig,  obtained  at  a  time  when  the 
primary  destruction  of  the  bacteria  has  been  followed  by  their 
reappearance  in  encapsulated  form,  to  a  mixture  of  normal  serum 
and  leukocytes,  in  certain  definite  proportions,  it  can  be  shown  that 
the  bactericidal  effect  of  the  leukins  is  completely  suspended.  If 
the  cells  contained  in  this  mixture  are,  however,  killed  and  simul- 
taneously extracted  by  alternate  freezing  and  heating  to  56°  C., 
the  resultant  solution  is  again  bactericidal,  showing  that  the  active 
substances  are  not  injured  by  the  aggressin  exudate,  but  that  their 
formation  is  merely  impeded.  The  reason,  then,  why  the  cap- 
sulated  organisms  can  at  first  develop  in  the  body  of  a  fresh  animal 
is  to  be  sought  in  the  primary  absence  of  aggressins,  which  only 
develop  in  sufficient  quantity  after  a  certain  length  of  time. 

When  this  point  has  been  reached,  the  animal  is  void  of  all  defen- 


84          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

sive  measures,  as  the  capsulated  organisms  which  alone  are  present 
are  not  susceptible  to  phagocytosis,  and  as  bactericidal  substances 
are  no  longer  formed,  owing  to  the  paralyzing  effect  of  the  aggressin 
upon  the  leukocytes,  so  that  boundless  multiplication  and  a  general 
invasion  of  the  body  are  the  outcome. 

If  the  infection  of  the  guinea-pig  is  started  subcutaneously  instead 
of  intraperitoneally  the  picture  is  somewhat  different.  In  this  case 
a  primary  destruction  of  the  organisms,  comparable  to  what  occurs 
in  the  peritoneal  cavity,  is  not  seen;  on  the  contrary,  there  is  active 
multiplication  from  the  start.  The  explanation  of  this  difference  is 
no  doubt  to  be  sought  in  the  greater  difficulties  which  would  present 
themselves  to  a  prompt  collection  of  cells  and  serum  at  the  point  of 
attack. 

In  either  event  the  infection,  when  once  it  has  started,  progresses 
without  resistance  and  ultimately  leads  to  the  death  of  the  animal. 
How  this  is  brought  about  is  unknown.  So  much,  however,  seems 
to  be  certain  that  unlike  the  infections  with  the  so-called  necropara- 
sites  (tetanus,  diphtheria,  botulismus)  toxins  do  not  play  a  role  in 
anthrax,  and  we  can  accordingly  only  say  that  the  fatal  end  in  infec- 
tions of  this  order  must  result  in  an  indirect  way.  Significant  in 
this  connection  is  the  fact  that  anthrax  infection  in  animals  that 
are  naturally  somewhat  resistant,  or  in  others  in  which  a  certain 
degree  of  resistance  has  been  artificially  produced,  is  followed  by 
symptoms  of  actual  disease  and  a  gradual  decline  in  health  until 
death  ultimately  occurs. 

Similar  considerations  apply  to  infections  with  streptococci  and 
possibly  also  with  pneumococci.  While  culture  streptococci  readily 
succumb  to  phagocytosis,  the  animalized  organism  is  highly  re- 
sistant. But  while  the  anthrax  bacillus  (in  the  absence  of  aggres- 
sins)  is  readily  destroyed  by  living  leukocytes  aphagocitically  (i.  e., 
without  phagocytosis,  through  the  agency  of  the  liberated  leukins), 
this  does  not  occur  in  the  case  of  the  streptococcus.  Against  this 
organism  the  body  apparently  possesses  no  defence  excepting  the 
phagocytic  function  of  the  leukocytes,  and  this  the  truly  infectious 
streptococcus  can  overcome  only  too  readily  through  the  agency  of 
its  aggressins.  The  importance  of  the  latter  will  be  appreciated,  if 
we  bear  in  mind  that  a  streptococcus  exudate,  which  has  been 
rendered  cell-  and  bacterium-free  by  centrifugation,  is  capable,  in 
suitable  quantity,  of  completely  inhibiting  the  bacteriotropins  of  a 


MECHANISM  IN  INFECTIONS  WITH  SEMIPARASITES     85 

corresponding  immune  serum  and  of  thus  preventing  phagocytosis 
and  bacterial  destruction  through  this  agency. 

In  this  respect  there  is  a  striking  difference  between  the  truly 
infectious  and  the  non-infectious  or  merely  locally  infectious  strepto- 
coccus, for  on  adding  aggressin  from  an  infectious  to  a  non-infectious 
strain  the  latter  is  not  protected  against  phagocytosis. 

Offensive-defensive  Mechanism  in  Infections  with  Semiparasites.— 
If  now  we  turn  out  attention  to  the  offensive-defensive  mechanism 
which  is  thrown  into  operation  in  infections  with  the  so-called  semi- 
parasites,  of  which  the  typhoid  bacillus  and  the  cholera  vibrio  are 
typical  examples,  we  meet  with  still  a  different  picture,  which  is 
fairly  well  defined  also,  although  it  has  not  been  worked  out  in  its 
details  so  thoroughly  as  we  have  seen  it  in  anthrax.  A  great  deal 
again  depends  upon  the  quantitative  relations  at  the  point  of  infec- 
tion. If  the  infecting  dose  (of  the  cholera  vibrio,  for  example,  given 
intraperitoneally)  is  large,  e.  g.,  several  multiples  of  the  quantity 
which  will  just  produce  infection,  there  is  virtually  no  evidence  of  a 
defensive  reaction.  The  organisms  multiply  from  the  start,  or  at 
least  do  not  diminish  in  number  even  during  the  first  few  hours; 
there  is  no  evidence  of  phagocytosis  or  of  extracellular  degeneration. 
Leukocytes  indeed  are  relatively  scant,  while  the  abdominal  cavity 
is  filled  with  a  serous  exudate,  in  which  the  bacteria  multiply  as  in 
an  ordinary  culture  medium.  The  animal  at  the  same  time  shows 
evident  signs  of  being  ill;  the  abdomen  is  tense  and  exceedingly 
tender,  the  hair  is  ruffled,  the  temperature  drops,  and  death  soon 
results. 

From  such  a  picture  one  would  be  led  to  conclude  that  the  animal 
was  devoid  of  all  defensive  means  against  the  organism  in  question. 
This,  however,  would  be  erroneous,  for  on  injecting  another  guinea- 
pig  with  a  much  smaller  dose,  e.  g.,  one-half  the  minimal  infecting 
dose,  which  after  all  represents  an  enormous  number  of  bacteria, 
the  findings  will  be  altogether  different.  If  specimens  of  the  peri- 
toneal contents  are  removed  at  various  intervals  after  the  injection, 
it  will  be  observed  at  a  very  early  period  that  active  bacteriolysis 
is  already  going  on  which  may  indeed  be  so  extensive  that  after  one 
hour  the  peritoneal  cavity  may  have  become  microscopically  free  of 
organisms.  But  even  if  this  does  not  result,  the  destruction  of 
bacteria  is  in  any  event  very  considerable,  and  becomes  complete 
through  the  introduction  of  a  new  factor,  viz.,  the  appearance  of 


86         THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

large  numbers  of  leukocytes  which  are  mainly  of  the  polynuclear 
neutrophilic  type.  These  dispose  of  the  remaining  organisms  by 
phagocytosis,  and  the  peritoneal  cavity  finally  becomes  sterile. 

This  means,  in  other  words,  that  the  animal  which  showed  no 
evidence  of  a  defensive  reaction  in  the  first  experiment,  actually 
had  a  very  definite  mechanism  of  this  kind  at  its  disposal,  and  the 
conclusion  is,  no  doubt,  justifiable  that  in  the  first  instance  the  dis- 
tribution of  the  normal  bactericidal  substances  of  the  serum  among 
the  enormous  number  of  bacteria  (or  its  exhaustion  by  relatively 
few  organisms)  was  insufficient  to  bring  about  any  recognizable 
effect,  and  its  renewed  production,  if,  indeed,  this  occurred  at  all, 
was  too  small  or  delayed  too  long  to  cause  any  material  retardation 
of  the  final  outcome. 

The  appearance  of  the  second  line  of  defence,  viz.,  the  leukocytes, 
was  evidently  also  delayed  too  long,  if,  indeed,  we  are  permitted  to 
speak  of  a  delay  at  all  under  such  conditions  where  there  is  evidence, 
both  experimental  and  clinical,  to  show  that  in  infections  with  over- 
whelming numbers  of  organisms  the  leukocytic  mobilization  may  be 
arrested  almost  altogether. 

If  we  compare  the  picture  illustrated  by  the  second  experiment 
with  what  we  have  seen  in  the  corresponding  anthrax  experiment 
there  is  a  certain  resemblance,  for  here  as  there  the  peritoneal  cavity 
is  virtually  freed  from  bacteria  soon  after  the  primary  invasion;  but 
while  in  infections  with  the  semiparasites  or  at  least  with  organisms 
of  the  type  of  the  typhoid  and  cholera  bacilli,  the  organisms  remain 
absent,  or  become  so  (unless  too  large  a  dose  had  been  chosen)  in 
anthrax  there  is  invariably  a  second  phase  which  is  characterized 
by  the  return  of  the  germs  and  their  subsequent  multiplication 
without  further  hindrance,  even  when  a  small  dose  has  been  injected. 

Another  point  of  difference  also  exists  which  is  important,  for 
whereas  in  the  anthrax  experiment  the  primary  bactericidal  effect 
was  due  to  an  associated  phagocytic  and  aphagocytic  activity  of 
the  leukocytes  (in  the  presence  of  serum),  the  primary  destruction 
of  the  cholera  vibrios  was  essentially  brought  about  by  the  normal 
bacteriolysins  of  the  serum.  Whether  during  the  second  phase 
of  the  cholera  experiment,  when  the  leukocytes  appear,  a  leukin 
action  also  takes  place,  seems  doubtful.  If  it  occurs  it  certainly 
plays  a  relatively  insignificant  role.  Then,  again,  while  animaliza- 
tion  (encapsulation)  of  the  anthrax  bacilli  leads  to  successful  resist- 


MECHANISM  IN  INFECTIONS  WITH  SEMIPARASITES       87 

ance  against  phagocytosis,  the  corresponding  changes  which  take 
place  in  the  cholera  vibrio  and  the  typhoid  bacillus  and  which 
are  represented  by  an  hypertrophy  of  the  ectoderm,  do  not  lead 
to  the  same  degree  of  protection. 

Evidently,  then,  there  is  a  marked  difference  in  the  character  of 
the  strife  between  the  defensive  forces  of  the  guinea-pig  and  the 
two  types  of  organisms.  On  the  one  hand,  the  anthrax  bacillus 
gains  the  upper  hand  through  its  successful  resistance  to  phagocy- 
tosis and  the  inhibitory  effect  of  its  aggressins  upon  the  production 
of  leukins,  even  though  the  appearance  of  the  leukocytes  at  the 
point  of  infection  is  not  seriously  impeded.  In  infections  with  the 
semiparasites,  on  the  other  hand,  the  organisms  conquer  essen- 
tially through  the  negatively  chemotactic  effect  of  their  aggressions 
upon  the  cells,  which  are  thus  kept  at  a  distance  and  through  the 
resistance  of  the  animalized  individuals  to  the  ordinary  bacterio- 
lytic  influences  of  the  serum. 

Between  the  two  extremes  which  have  been  represented  above, 
i.  e.,  the  effect  following  the  injection  of  large  and  of  subinfectious 
doses  of  the  cholera  vibrio  every  possible  gradation  is  possible  and 
can  actually  be  reproduced  in  the  animal  experiment.  If  thus  the 
minimal  infecting  dose  is  injected  there  will  usually  be  a  primary 
bacteriolysis  of  considerable  extent,  which  then  gives  way  to  a 
gradually  developing  increase  in  the  number  of  the  organisms.  At 
first,  as  the  leukocytes  begin  to  appear,  this  proceeds  slowly,  but 
after  a  little  while  the  organisms  definitely  secure  the  upper  hand 
and  coincidently  the  further  influx  of  cells  is  more  or  less  completely 
arrested  and  the  disease  pursues  its  course  to  a  fatal  termination. 

That  the  leukocytic  insufficiency  is  really  the  deciding  factor  in 
the  victory  of  the  bacteria  in  such  a  case  can  be  very  well  shown 
by  previously  injecting  the  animal  with  the  leukocytes  of  a  second 
one  and  then  introducing  the  minimal  dose  of  bacteria  which  in 
the  untreated  animal  would  invariably  produce  a  fatal  result.  It 
will  now  be  seen  that  the  animal  does  not  succumb,  and  if  a  series 
of  corresponding  experiments  be  carried  out  it  can  be  demonstrated 
that  a  number  of  multiples  of  the  originally  just  infecting  dose 
must  be  injected  in  order  to  kill.  Weil  has  shown  very  satisfactorily 
that  this  result  is  purely  referable  to  the  action  of  the  leukocytes 
and  not  to  any  bacteriolysins  that  may  be  present,  by  previously 
rendering  the  latter  inactive  with  so-called  complement-binding 


88          THE  BACTERICIDAL  SUBSTANCES  OF  THE  BLOOD 

substances,  when  infection  in  the  untreated  animal  may  be  brought 
about  with  subminimal  infecting  doses  (as  compared  with  a  con- 
trol animal),  while  in  one  that  has  been  previously  rendered  hyper- 
leukocytic  this  effect  is  not  obtained. 

Upon  the  basis  of  analytical  studies  such  as  those  outlined  in 
the  foregoing  pages,  incomplete  as  they  are,  we  can  now  distinguish 
three  different  types  of  infection  (excluding  those  with  the  so-called 
necroparasites,  in  which  a  successful  infection  can  scarcely  be 
brought  about  under  ordinary  conditions).  In  the  first,  represented 
by  the  anthrax  bacillus,  the  serum  in  itself  is  either  inactive  or 
shows  but  slight  inhibitory  qualities,  while  the  combination  of 
serum  with  leukocytes  has  strong  antibacterial  properties  which 
can  be  completely  overcome,  however,  through  the  aggressivity  of 
the  organism. 

The  second  type  is  represented  by  various  streptococci,  staphylo- 
cocci  and  certain  vibrios  (el  Tor.),  i.  e.,  organisms  which  stand  very 
close  to  the  group  of  the  true  parasites.  In  such  infections  the 
serum  alone  manifests  but  little  bactericidal  effect,  while  the  anti- 
bacterial action  of  the  serum,  when  combined  with  leukocytes, 
is  strongly  marked  and  can  be  only  partially  overcome  by  the 
aggressivity  of  the  organism. 

In  the  third  type  the  serum  alone,  as  well  as  in  combination  with 
leukocytes,  shows  marked  antibacterial  properties,  the  former  by 
itself  being  sometimes  sufficient  to  offset  the  aggressivity  of  the 
corresponding  bacteria.  The  animalization  of  the  organisms  is  here 
of  little  avail,  as  a  protective  measure  against  phagocytosis,  while 
it  is  partially  effective  in  the  case  of  the  serum.  Most  members 
of  the  typhoid  and  the  vibrio  group  fall  under  this  category. 


CHAPTER   VII 
ANTIGENS  AND  ANTIBODIES 

WE  have  seen  in  the  foregoing  chapters  that  the  normal  animal 
has  a  defensive  mechanism  at  its  disposal  with  which  it  may  suc- 
cessfully meet  a  developing  infection,  with  certain  organisms  at 
least,  providing  that  the  invading  numbers  are  not  too  large.  In 
laboratory  parlance  we  express  this  by  saying  that  successful  resist- 
ance is  possible,  if  the  bacterial  dose  falls  short  of  the  minimal 
infecting  amount,  or  if  this  should  be  exceeded,  at  least  of  the 
minimal  fatal  amount.1 

If  now  we  compare  the  bacteriolytic  titer  of  the  serum  of  an  animal 
that  has  received  an  injection  of  a  subfatal  dose  with  that  of  a 
normal  control,  or  with  that  which  the  same  animal  showed  before 
the  injection,  a  remarkable  increase  will  be  noted  which  may  be 
further  raised  by  additional  injections.  Upon  then  examining  the 
peritoneal  contents  of  a  normal  animal  that  has  received  a  minimal 
fatal  dose  and  comparing  the  results  with  the  findings  in  a  second 
animal  which  has  been  previously  injected  with  a  subfatal  dose  and 
which  now  receives  the  same  amount  as  the  first,  it  will  be  noted 
that  at  a  certain  time  the  peritoneal  fluid  of  the  previously  injected 
animal  will  have  become  sterile,  while  that  of  the  untreated  control 
is  swarming  with  organisms;  and,  moreover,  while  the  latter  dies, 
the  other  recovers  and  thus  shows  itself,  relatively  at  least,  immune, 
using  this  term  in  the  original  sense  of  its  meaning  and  synony- 
mously with  "resistant." 

This  immunity  was  evidently  produced  through  the  activity  of 
the  animal  itself,  and  is  hence  appropriately  spoken  of  as  active 
immunity  in  contradistinction  to  passive  immunity,  which  latter 
results  when  the  immunity-bestowing  substances  that  were  actively 
produced  in  the  one  animal  are  artificially  transferred  to  a  second 
(normal)  one.  The  possibility  of  such  a  transference  can  be  readily 

1  These  considerations  apply  essentially  to  infections  with  the  so-called  semi- 
parasites,  exemplified  by  the  cholera  vibrio  and  the  typhoid  bacillus. 


90  ANTIGENS  AND  ANTIBODIES 

demonstrated  by  injecting  a  normal  animal  with  a  minimal  fatal 
dose  of  the  corresponding  bacteria  together  with  an  appropriate 
quantity  of  serum  obtained  from  an  "immunized"  animal.  In 
such  an  event  death  does  not  result,  because  the  animal  has  here 
been  passively  immunized  by  the  serum  of  the  immune  animal, 
and  now  in  turn  develops  an  active  immunity  as  the  result  of  the 
introduction  of  the  bacteria. 

In  a  previous  chapter  we  have  seen  that  the  bacteriolytic  action 
of  normal  serum  is  referable  to  the  associated  activity  of  two  sub- 
stances, viz.,  the  thermolabile  complement  and  the  thermostabile 
amboceptor.  On  studying  a  bacteriolytic  immune  serum  in  this 
direction,  it  may  be  shown  that  here  also  the  destructive  action  upon 
the  bacteria  is  dependent  upon  complement  and  corresponding 
amboceptor,  and  that  its  greater  degree  of  activity  as  compared 
with  normal  serum  is  altogether  owing  to  an  increased  content  of 
the  latter. 

At  a  time  when  the  antibacterial  action  of  the  normal  blood  serum 
was  first  discovered  the  question  of  the  origin  of  the  "alexins"  was 
wrapped  in  complete  obscurity.  In  view  of  the  manner  in  which 
the  production  of  the  immune  amboceptors  takes  place  there  can 
be  no  doubt  that  a  direct  connection  exists  between  their  appear- 
ance and  the  introduction  of  the  corresponding  bacteria,  and  upon 
injecting  different  animals  with  different  species  of  bacteria  we  obtain 
evidence  of  a  most  remarkable  specificity  in  the  nature  of  the  response, 
which  one  can  well  compare  to  the  vibratory  response  which  is  called 
forth  in  tuning  forks  of  different  pitch  by  striking  forks  of  corre- 
sponding pitch. 

Further  studies  in  this  direction  have  shown  that  the  appearance 
of  such  immune  amboceptors  takes  place  according  to  a  fairly 
definite  rule :  immediately  following  the  injection  a  period  of  latency 
can  thus  be  observed  which  lasts  for  a  few  days  and  is  then  followed 
by  a  critical  ascent  of  the  curve  leading  to  a  maximal  point  from 
which  there  is  in  turn  a  corresponding  drop  which  at  first  is  fairly 
abrupt  and  later  more  gradual,  and  hence  a  slow  return  to  previously 
existing  conditions.  As  the  same  result  is  obtained  after  the  injec- 
tion of  dead  bacteria  it  is  clear  that  the  prolonged  effect  which 
follows  the  introduction  of  the  organisms  cannot  be  referable  to  pos- 
sible variations  in  their  number  which  one  might  otherwise  imagine 
to  be  operative  on  different  days  and  at  different  hours,  nor  can  the 


ALLERG1A  91 

cessation  in  the  formation  of  the  amboceptors  be  explained  on  the 
basis  of  the  gradual  disappearance  of  the  bacteria. 

On  the  contrary,  it  is  evident  that  a  stimulus  has  been  given  which 
remains  operative  long  after  the  primary  impulse  to  amboceptor 
formation  has  ceased;  to  return  to  our  simile,  the  second  tuning 
fork  still  vibrates,  though  the  first  one  which  gave  rise  to  its  vibra- 
tion has  already  become  quiescent.  The  animal  has  coincidently 
developed  a  resistance  to  the  organisms  in  question  which  is  far 
beyond  its  original  value;  it  may  indeed  be  absolute  so  that  sub- 
sequent infection  is  altogether  impossible.  This  resistance,  more- 
over, in  the  case  of  some  organisms  at  least,  may  be  lasting,  e.  g., 
the  immunity  which  follows  an  attack  of  typhoid  fever  or  of 
Asiatic  cholera  in  man. 

If  the  blood  of  a  recently  injected  animal  (using  the  typhoid 
bacillus  for  example)  is  further  examined  it  will  be  found  that,  aside 
from  the  resultant  bacteriolytic  properties,  it  has  developed  still 
other  characteristics  which  the  serum  of  the  untreated  animal 
either  did  not  possess  at  all,  or  if  so,  only  to  a  slight  extent.  For 
it  will  be  observed  that  such  blood,  even  though  freely  diluted,  has 
now  the  power  of  causing  the  arrest  of  motility  and  the  clumping 
or  agglutination  of  the  corresponding  organisms  (Widal  reaction), 
and  this  result,  like  the  production  of  the  bacteriolysins,  is  not 
dependent  upon  the  introduction  of  living  bacteria,but  may  be  effected 
with  dead  organisms,  as  well.  If,  further,  analogous  experiments 
are  carried  out  writh  organisms  like  the  diphtheria  or  the  tetanus 
bacillus  it  will  be  observed  that  still  other  changes  develop  in  the 
body  of  the  infected  animal  and  that  bodies  here  appear  in  the  blood 
serum  which  have  the  power  of  neutralizing  the  specific  poisons 
formed  by  the  organisms  in  question.  Then,  again,  a  curious  reac- 
tion develops  in  animals  which  have  been  infected  with  the  tubercle 
bacillus,  for  example,  for  on  subsequent  injection  with  certain  deriva- 
tives of  this  organism  (tuberculin)  the  animal  responds  with  fever 
while  the  previously  untreated  control  shows  ho  reaction  whatever. 

Allergia. — These  various  responses  in  the  reaction  of  the  animal 
to  the  introduction  of  bacteria  are  now  recognized  as  being  merely 
a  partial  expression  of  a  general  biological  law^,  to  wit,  that  the 
animal  organism  invariably  responds  to  the  parenteral  introduction 
of  foreign  cells  (i.  e.,  the  introduction  of  cells  by  other  channels 
than  through  the  gastro-intestinal  canal,  whether  these  be  of  animal 


92  ANTIGENS  AND  ANTIBODIES 

or  vegetable  nature),  or  of  the  products  of  foreign  cells,  insofar  at 
least  as  they  are  of  protein  character,  by  the  production  of  substances 
which  in  a  general  way  tend  to  antagonize  or  even  to  destroy  those 
which  indirectly  gave  rise  to  their  formation.  For  this  altered 
behavior  of  the  "treated"  as  compared  with  the  "non-treated" 
animal,  v.  Pirquet  has  proposed  the  very  appropriate  and  at  the 
same  time  non-committal  term  allergia  (dtty  Iftfeta),  which  merely 
denotes  a  state  of  altered  power  of  reaction  on  the  part  of  the 
"treated"  organism. 

The  reaction  products  which  are  formed  in  the  body  of  the  treated 
animal  are  conjointly  spoken  of  as  antibodies,  and  the  substances 
whose  introduction  from  without  give  rise  to  their  formation  are 
similarly  termed  antigens  or  allergens. 

The  discovery  of  these  substances  and  their  bearing  upon  the 
subject  of  immunity  has  opened  up  an  enormous  field  for  fruitful 
research,  not  only  in  the  domain  of  medical  science,  but  in  that  of 
general  biology  as  well,  and  has  already  led  to  results  which  the  boldest 
flight  of  the  imagination  would  not  have  thought  possible  twenty- 
five  years  ago.  The  earliest  and,  in  a  manner,  the  most  brilliant 
investigations  in  this  direction  we  owe  to  the  genius  of  Behring 
and  his  collaborators,  Wernicke  and  Kitasato. 

Antitoxins. — These  investigators  found  that  the  serum  of  animals 
which  had  been  rendered  immune  to  the  specific  toxins  of  tetanus 
and  diphtheria  had  acquired  the  power  of  neutralizing  the  harmful 
effect  of  those  poisons,  and  Tizzoni  and  Catani  introduced  the  term 
antitoxin  to  denote  the  substance  to  which  this  action  is  due.  Here 
the  way  was  shown  for  the  first  time  along  which  it  would  be  pos- 
sible successfully  to  combat  one  of  the  most  common  and  most 
dangerous  diseases  which  has  threatened  the  human  race  since  time 
immemorial.  Scarcely  twenty-five  years  have  now  passed  since 
Bearing's  announcement  to  the  world  (1890)  that  it  is  not  only 
possible  to  protect  the  human  being  against  infection  with  the  diph- 
theria bacillus,  but  that  the  disease  may  be  arrested  even  after  it 
has  gained  a  foothold — and  all  this  through  the  injection  of  a  rela- 
tively small  amount  of  serum  derived  from  a  horse  that  has  been 
previously  treated  with  a  culture  of  diphtheria  bacilli  or  their 
specific  toxin.  How  well  Behring's  discovery  has  served  the 
human  race  is  already  a  matter  of  history. 

For  a  while  hope  ran  high  that  it  would  only  be  a  matter  of  time 


ANTITOXINS  93 

before  equally  efficacious  antitoxins  would  be  discovered  for  the 
treatment  of  all  the  other  bacterial  infections  to  which  both  man 
and  beast  are  prone,  but  this  was  soon  doomed  to  disappointment. 
Why  this  should  be  is  now  fairly  clear  to  us,  since  we  have  become 
familiar  with  the  offensive  mechanism  through  which  the  foreign 
organism  seeks  to  maintain  itself  in  the  animal  body,  and  through 
which  the  destruction  of  the  host  may  even  be  accomplished.  We 
have  thus  seen  that  both  the  diphtheria  and  the  tetanus  bacillus 
are  organisms  of  the  lowest  grade  of  infectiousness  which  cannot 
possibly  maintain  themselves  in  normal  tissues  and  are  readily 
and  rapidly  destroyed  through  the  activity  of  both  serum  and  cells, 
but  which  kill  nevertheless  through  the  wonderful  activity  of  their 
specific  poisons.  Against  these  the  normal  organism  either  possesses 
no  antitoxin  at  all  or  such  small  amounts  that  a  fatal  end  only  too 
often  occurs  even  though  the  infection,  as  such,  has  been  or  is  being 
successfully  combated.  As  a  result  of  the  infection,  an  attempt 
at  antibody  (antitoxin)  formation  is,  of  course,  made  (active  immuni- 
zation), but  unfortunately  the  toxin  may  be  able  to  produce  its 
harmful  effect  before  enough  antitoxin  is  formed  to  neutralize  its 
action.  That  under  such  circumstances  the  introduction  of  anti- 
toxin from  without  (passive  immunization)  is  the  logical  method  of 
treatment,  goes  without  saying. 

In  other  infections  the  conditions  are  different.  Unfortunately, 
the  majority  of  organisms  which  are  pathogenic  for  man  are  either 
not  true  toxin  producers  at  all,  or,  if  so,  their  infectiousness  is  of  a 
much  higher  order,  so  that  the  mere  introduction  of  an  antitoxin, 
even  though  it  were  tuned  to  the  corresponding  toxin,  so  to  speak, 
would  not  suffice  to  bring  the  disease  resulting  from  the  infection 
to  a  standstill.  What  is  needed  in  such  cases  is  something  that  will 
prevent  the  continuance  of  the  infection,  and  that  something  can 
scarcely  be  of  the  nature  of  an  antitoxin. 

Aside  from  diphtheria  and  tetanus,  there  is  actually  only  one 
organism,  infection  with  which  lends  itself  to  antitoxin  treatment, 
pure  and  simple,  namely,  the  bacillus  botulinus.  Of  the  other  patho- 
genic organisms  the  bacillus  pyocyaneus,  the  staphylococcus,  the 
typhoid,  paratyphoid,  and  dysentery  bacillus,  the  vibrio  of  cholera 
Asiatica  and  related  organisms,  the  plague  bacillus,  and  the  bacillus 
of  symptomatic  anthrax  are  known  or  supposed  to  form  true  toxins 
even  though  to  a  limited  extent  only;  but  for  the  reasons  just 


94  ANTIGENS  AND  ANTIBODIES 

indicated  the  corresponding  antitoxic  sera  are  of  little  avail  in  the 
treatment  of  the  corresponding  maladies. 

Interesting  from  theoretical  grounds  is  the  fact  that  many  other 
true  toxins  have  been  discovered  which  are  not  of  bacterial  origin. 
To  this  category  belong  certain  snake  poisons  (venin),  the  phryn- 
olysin  found  in  the  skin  glands  of  toads  (Bombinator  igneus)  and 
salamanders  (Sieboldia),  a  poison  obtained  from  special  glands  of 
certain  fishes  (Trachinus),  the  arachnolysin  of  various  spiders  (Latro- 
dectes  and  Epeira),  the  poison  of  wasps  and  bees,  the  ichthyotoxin 
which  is  found  in  the  serum  of  the  eel,  and  possibly  also  the  toxin 
producing  fatigue,  which,  according  to  Weichardt,  is  formed  in 
the  muscles  after  severe  exercise  (kenotoxin) .  In  addition  to  these, 
certain  toxins  produced  by  higher  plants  are  recognized  as  possessing 
true  antigenic  properties,  such  as  the  ricin  obtained  from  the  seeds 
of  the  castor  oil  bean  (Ricinus  communis),  the  abrin  of  the  jequirity 
bean  (Abrus  precatorius),  the  crotin  of  croton  seeds  (Croton  tiglium), 
the  robin  obtained  from  the  bark  of  the  Robinia  pseudacacia,  and 
the  phallin  of  the  poisonous  mushroom  Amanita  phalloides. 

All  these  substances  are  characterized  by  their  poisonous  nature 
and  the  fact  that  their  introduction  into  the  animal  organism,  in 
suitable  dosage,  gives  rise  to  the  production  of  corresponding  anti- 
toxins which  in  turn  have  the  power  of  neutralizing  the  toxic  effect 
of  the  substances  that  gave  rise  to  their  formation. 

Bacteriolysins. — Closely  following  upon  the  discovery  of  the  anti- 
toxins came  the  work  of  Pfeiffer  and  his  pupils  on  the  bacteriolysins 
(1894),  viz.,  antibodies  which  result  upon  immunization  (vaccination) 
with  various  bacteria  and  which  possess  the  property  of  causing  the 
dissolution  of  the  corresponding  organisms  (Pfeiffer's  phenomenon). 
Antibodies  of  this  order  are  notably  produced  against  certain  vibrios, 
such  as  the  vibrio  of  cholera  Asiatica,  the  vibrio  Metschinkoffi  and 
related  forms,  against  the  typhoid  and  paratyphoid  bacillus,  the 
colon  bacillus,  the  dysentery  bacillus,  the  bacillus  pyocyaneus,  the 
influenza  bacillus,  and  the  bacillus  of  bubonic  plague. 

When  these  substances  were  first  discovered  it  was  hoped  that  the 
corresponding  bacteriolytic  sera  would  be  found  to  possess  curative 
properties  analogous  to  those  of  the  antitoxic  sera,  but  it  was  soon 
ascertained  that  while  they  can  prevent  infection  when  they  are 
introduced  together  with  the  organisms  or  shortly  after,  they  are  of 
little  if  any  apparent  avail  in  combating  an  already  established 


AGGLUTININS  95 

infection.  Why  this  should  be  is  not  clear,  unless  we  assume  that 
the  organisms  have  developed  new  characteristics,  in  consequence  of 
which  they  are  no  longer  open  to  attack  by  the  bacteriolysins  of  the 
serum,  and  that  the  subsequent  defence  of  the  body  must  be  carried 
on  by  other  forces.  For  the  correctness  of  this  view  there  is  some 
actual  basis  (see  preceding  chapter),  but  even  so  the  last  word  on 
the  use  of  the  bacteriolytic  sera  has  probably  not  yet  been  spoken. 

But  in  any  event  the  discovery  of  the  bacteriolysins  must  be 
regarded  as  one  of  the  greatest  importance,  as  it  has  enabled  us  to 
gain  a  certain  insight  into  the  defensive  mechanism  of  the  animal 
body,  which  is  most  essential  to  further  advance.  Practically 
important  is  the  fact  that  the  action  of  the  bacteriolytic  immune 
amboceptors  is  specific  and  thus  permits  of  a  twofold  diagnostic 
application.  As  the  amboceptor  content  of  the  immunized  animal 
is  always  higher  than  that  of  the  normal  control,  a  higher  titer  in 
reference  to  a  given  organism  may  be  regarded  as  evidence  of  a 
preceding  infection.  Similarly  one  can  use  an  immune  serum  for 
the  purpose  of  identifying  a  given  organism  by  comparing  its  action 
with  that  of  a  normal  serum  upon  the  organism  in  question,  in  the 
peritoneal  cavity  of  a  guinea-pig.  Both  methods  are  in  actual  use, 
the  first  for  ascertaining  whether  or  not  an  individual  has  recently 
passed  through  an  attack  of  cholera,  the  other  for  establishing  the 
identity  of  the  corresponding  organism  after  its  isolation  from  the 
feces.  (For  a  description  of  the  method  see  Diagnostic  Bacteriolytic 
Reactions.) 

Agglutinins. — The  next  group  of  antibodies  was  discovered  by 
Gruber  and  Durham  (1896).  These  are  termed  agglutinins  from  the 
fact  that  the  sera  in  question,  when  brought  together  with  emulsions 
of  the  corresponding  organisms,  will  cause  the  "clumping"  or  agglu- 
tination of  the  bacteria,  and  if  these  are  normally  motile,  incidentally 
affect  their  loss  of  motility.  As  this  property  also  is  specific  within 
certain  limitations  and  the  technique  involved  in  its  demonstration 
very  simple,  the  principle  has  been  extensively  utilized  for  diagnostic 
purposes.  As  in  the  case  of  the  bacteriolysins  it  may  be  applied 
both  for  the  identification  of  a  given  organism  and  in  search  for  the 
corresponding  agglutinin.  Under -the  name  of  the  Widal  reaction  the 
test  is  now  used  the  world  over  as  one  of  the  most  importantjactors 
in  the  diagnosis  of  typhoid  fever  (see  Agglutination  Reaction). 

The  significance  of  the  process  of  agglutination  is  not  very  clear. 


96  ANTIGENS  AND  ANTIBODIES 

That  the  life  of  the  organism  in  itself  has  nothing  to  do  with  the 
production  of  the  phenomenon  is  proved  by  the  fact  that  the  same 
result  is  brought  about  with  dead  cultures.  On  the  other  hand  it 
can  be  shown  that  the  process  of  agglutination  does  not  lead  to  the- 
destruction  of  the  bacteria;  these  may,  in  fact,  multiply  in  the  agglu- 
tinated state.  Under  certain  conditions  they  will  then  grow  out  in 
threads  which  are  twisted  upon  themselves  so  as  to  form  complicated 
skeins — a  behavior  which  was  first  noted  by  Pfaundler  and  which  is 
spoken  of  as  Pfaundler's  Fadenreaktion  (thread  reaction). 

Gruber,  Durham,  Baumgarten  a.  o.,  at  first  looked  upon  the 
agglutinins  as  being  identical  with  the  bacteriolysins,  the  process 
of  agglutination  being  interpreted  as  a  stage  preparatory  to  bacte- 
riolysis. From  this  standpoint  their  formation  could  be  viewed  as 
evidence  of  a  protective  reaction  on  the  part  of  the  animal  body. 
Subsequent  investigations,  however,  have  rendered  this  position 
untenable.  Cholera  immune  serum  thus  loses  its  agglutinating 
properties  after  a  certain  length  of  time,  even  though  its  bacteriolytic 
power  remains  in  full  activity.  Then,  again,  it  has  been  observed 
that  in  typhoid  fever  the  agglutinative  and  the  bactericidal  power 
of  the  patient's  serum  do  not  necessarily  run  a  parallel  course,  but 
may  actually  diverge.  Gengou  further  showed  that  the  agglutinins 
do  not  dialyze  through  collodium,  while  the  lysins  do,  and  that  the 
injection  of  sodium  carbonate  increases  the  bactericidal,  but  not  the 
agglutinative  power.  While  the  agglutinins  are  thus  unquestionably 
not  identical  with  the  bacteriolysins  there  are  reasons  for  believing 
that  they  may  after  all  not  be  antibodies  sui  generis  (see  Precipi- 
tins). 

The  most  important  organisms  with  which  agglutinin  formation 
has  been  successfully  produced  are  the  typhoid  and  paratyphoid 
(A  and  B),  the  cholera  and  dysentery  bacillus,  the  bacillus  lacti 
aerogenes,  the  diphtheria  bacillus,  the  tubercle  bacillus,  the  plague 
bacillus,  the  bacillus  of  glanders,  the  influenza  bacillus,  Friedlander's 
bacillus,  the  bacillus  of  tetanus  and  of  rhinoscleroma,  the  pyocyaneus 
and  proteus  bacillus,  the  bacillus  enteritidis,  the  cholera  vibrio, 
the  micrococcus  melitensis,  staphylococcus  aureus,  streptococcus 
pyogenes,  the  pneumococcus,  and  the  meningococcus  intracellularis. 

Precipitins. — Shortly  after  the  discovery  of  the  agglutinating  power 
of  certain  antisera,  Kraus  ascertained  that  such  sera  when  brought 
together  with  the  clear  filtrates  of  the  corresponding  bouillon  cultures 


CYTOLYSINS  97 

will  cause  the  appearance  of  a  turbidity  which  gradually  collects 
at  the  bottom  of  the  tube  as  a  precipitate  (1897).  Further  studies 
then  showed  that  this  peculiar  behavior  is  owing  to  the  presence  of 
definite  antibodies  in  the  sera  of  the  injected  animals,  and  that  such 
antibodies  are  formed  whenever  foreign  albumins  either  of  animal 
or  vegetable  origin  are  introduced  through  parenteral  channels. 
From  their  precipitating  properties  these  substances  have  been 
termed  precipitins,  while  the  corresponding  antigen  is  termed 
precipitinogen. 

Like  the  bacteriolysins,  the  precipitins  have  been  shown  to  be 
specific  in  their  action,  within  certain  limitations  at  least,  and  the 
reaction  has  accordingly  been  used  for  the  purpose  of  identifying 
the  origin  of  various  albumins.  In  the  form  of  the  biological  blood 
test  the  principle  is  now  generally  utilized  for  the  purpose  of  deter- 
mining the  origin  of  blood  stains,  and  upon  the  same  basis  it  has 
been  possible  to  establish  zoological  relationship  between  different 
animals  (see  Precipitin  Test). 

Of  special  interest  is  the  fact  that  a  number  of  investigators  are 
now  inclined  to  regard  the  agglutinating  properties  of  the  various 
antisera  merely  as  one  form  of  expression  of  the  more  general  pre- 
cipitating qualities  of  the  same  sera,  so  that  according  to  this  con- 
ception the  agglutinins  as  antibodies  sui  generis  would  have  no 
existence.  It  is  supposed  that  agglutination  among  cells  corresponds 
exactly  to  agglutination  among  dissolved  albuminous  particles,  which 
latter  process  leads  to  what  we  are  accustomed  to  speak  of  as  pre- 
cipitation. This  view  is  supported  especially  by  Kraus,  v.  Pirquet, 
and  Wassermann.  These  observers  could  show  that  bacterial  filtrates 
are  capable  of  binding  agglutinin  and  that  the  filtrates  in  question 
must  hence  have  contained  agglutinable  substances.  This  is  well 
brought  out  if  cultural  filtrates  are  added  to  a  corresponding  agglu- 
tinating serum  in  sufficiently  large  quantity.  Under  such  conditions 
the  serum  may  lose  its  agglutinins  entirely.  If  insufficient  amounts 
of  filtrate  are  used,  on  the  other  hand,  the  agglutinating  titer  remains 
unaltered,  the  difference  in  behavior  being  explained  by  the  assump- 
tion that  much  smaller  quantities  of  precipitin  are  required  to  cause 
agglutination  than  to  bring  about  precipitation. 

Cytolysins. — Further  studies  of  the  peculiar  reaction  of  the  animal 
body  to  the  parenteral  introduction  of  foreign  cells  and  their  deriva- 
tives then  led  to  the  discovery  that  antibodies  of  amboceptor  type, 
7 


98  ANTIGENS  AND  ANTIBODIES 

i.  e.,  amboceptors  of  the  nature  of  the  bacteriolysins,  are  formed  not 
only  following  the  injection  of  bacteria,  but  also  upon  immunization 
with  other  cellular  elements,  using  the  term  immunization  in  the 
more  modern  sense  of  the  word,  viz.,  to  express  the  throwing  into 
action  of  the  remarkable  mechanism  which  results  in  the  develop- 
ment of  what  we  term  allergia,  and  of  which  the  formation  of 
antibodies  is  the  outcome  (see  above). 

Collectively  we  now  term  all  those  antibodies  of  amboceptor  type 
which  are  specifically  directed  against  animal  or  vegetable  cells, 
cytolysins  or  cytotoxins,  and  we  designate  the  individual  members  of 
this  order  according  to  the  cell  against  which  their  action  is  directed 
(sc.,  according  to  the  type  of  the  corresponding  antigen)  and  thus 
distinguish  between  erythrocytolysins  (hemolysins),  leukocytolysins 
(leuJcolysins) ,  epitheliolysins,  spermatolysins,  hepatolysins,  neurolysins, 
nephrolysins,  etc.  The  bacteriolysins  would  thus  merely  represent  a 
species  of  cytolysins. 

The  demonstration  of  some  of  these  antibodies  is  a  very  simple 
matter  as  their  action  in  reference  to  certain  cells  leads  to  alterations 
which  are  very  manifest,  while  with  others  we  rather  surmise  than 
are  able  to  prove  that  a  specific  effect  has  been  produced.  It  should 
be  mentioned,  moreover,  that  while  we  frequently  speak  of  these 
bodies  as  lysins,  a  true  dissolution  of  the  entire  cell  does  not  neces- 
sarily take  place,  and  it  would  really  be  more  appropriate  to  use 
the  synonymous  term  cytotoxin. 

The  first  cytolysins  of  animal  origin  to  be  discovered  were  the 
hemolysins  (1898).  After  Belfanti  and  Cortone  had  first  shown 
that  the  blood  of  an  animal  of  a  given  species  A  (horse),  which  had 
been  previously  injected  (immunized)  with  the  blood  of  an  animal 
of  a  different  species  B  (rabbit),  becomes  highly  toxic  for  all  represen- 
tatives of  species  B,  Bordet  demonstrated  that  this  result  is  accom- 
panied by  extensive  destruction  of  the  red  cells  in  the  animal  B.  He 
also  showed  that  the  same  effect  upon  the  red  cells  could  be  produced 
outside  of  the  body.  As  the  hemolyzing  power  of  the  serum  A  disap- 
pears after  heating  to  50°  to  60°  C.  for  about  thirty  minutes,  but  is 
restored  upon  the  addition  of  fresh  normal  serum  which  is  itself  non- 
hemolytic,  Bordet  concluded  that  the  hemolytic  effect  of  the  immune 
serum  depended  upon  the  joined  action  of  two  separate  bodies,  of 
which  one  is  present  in  every  fresh  normal  serum  and  is  thermo- 
labile,  while  the  other,  thermostabile  constituent,  is  formed  only 


CYTOLYSINS  99 

as  the  result  of  immunization,  and  is  hence  exclusively  found  in 
the  immune  serum. 

Ehrlich  and  Morgenroth  confirmed  these  findings  and  succeeded 
in  demonstrating  the  presence  of  both  components  in  the  fresh  serum 
of  the  immunized  animal.  These  two  substances,  as  we  have  already 
seen,  are  now  generally  spoken  of  as  amboceptor  (immune  body, 
Bordet's  substance  sensibilisatrice)  and  complement  (alexin  of  Buchner 
and  Bordet).  These  discoveries  were  of  fundamental  importance, 
as  they  immediately  led  to  the  recognition  that  the  bacteriolytic 
action  of  immune  sera  is  similarly  due  to  the  coaction  of  two  sub- 
stances, of  which  the  one  also  is  present  in  fresh  normal  serum,  while 
the  other  only  appears  as  the  result  of  immunization.  A  proper 
explanation  was  thus  given  for  Pfeiffer's  original  observation,  made 
in  1894,  that  inactivated  bacteriolytic  goat  serum  recovers  its 
bacteriolytic  action  when  introduced  into  the  peritoneal  cavity  of 
a  guinea-pig. 

Metschnikoff  and  Bordet  showed  that  the  same 'result  is  obtained 
by  mixing  such  inactivated  serum  with  fresh  peritoneal  fluid  in  vitro, 
or  by  adding  fresh  serum  or  freshly  defibrinated  blood,  the  reason, 
of  course,  being  that  under  the  conditions  of  the  experiment  the 
inactivated  immune  serum  finds  the  necessary  complement  both 
in  the  fresh  peritoneal  fluid  and  the  fresh  blood. 

Bordet  then  showed  that  while  naked  eye  observation  of  the 
process  of  hemolysis  suggests  that  the  red  cells  undergo  dissolution, 
this  is  in  reality  not  the  case,  but  that  the  hemoglobin  only  undergoes 
dissolution  into  the  outer  medium,  and  that  the  stromata  of  the 
cells  (the  shadows  of  the  red  cells)  can  be  separated  by  centrifugation, 
and  demonstrated  as  such. 

Similar  observations  were  made  regarding  the  action  of  the 
corresponding  cytotoxic  sera  upon  ciliated  epithelial  cells  and 
spermatozoa.  When  such  cells  are  introduced  into  the  peritoneal 
cavity  of  an  animal  that  has  been  correspondingly  immunized  the 
cells  promptly  lose  their  motility;  but  while  the  epithelial  cells 
gradually  undergo  destruction  the  spermatozoa  remain  unchanged. 

While  the  changes  which  are  thus  effected  by  cytotoxic  sera  in  the 
case  of  red  cells,  ciliated  epithelial  cells  and  spermatozoa  are  very 
apparent,  and  while  such  cells,  hence  represent  excellent  test  objects 
for  the  purpose  of  studying  the  nature  and  mode  of  action  of  the 
immune  sera  in  questipn,  the  demc-nstration^of  a  rceurotoxic,  hepato- 


100  ANTIGENS  AND  ANTIBODIES 

toxic,  or  nephrotoxic  influence  is  a  more  difficult  task.  Delezenne, 
it  is  true,  claims  to  have  obtained  hepatotoxic  sera  by  injecting  suit- 
able animals  with  dog's  liver,  and  states  that  such  sera  will  produce 
specific  impairment  of  the  liver  function  in  dogs,  as  evidenced  by 
diminished  elimination  of  urea,  by  increased  excretion  of  ammonia, 
and  the  appearance  of  leucin  and  tyrosin  in  the  urine,  and  that 
digestive  glucosuria  may  also  develop  when  the  animal  receives  an 
abundant  supply  of  sugar;  in  some  instances  death  even  occurred. 
The  same  writer  further  claims  to  have  obtained  a  neurotoxic  serum 
which  would  produce  vacuole  formation  and  chromatolysis  in 
ganglionic  cells.  Lindemann  and  Nefedieff  further  announced  that 
with  a  nephrotoxic  serum  they  could  bring  about  the  development 
of  albuminuria,  uremia,  and  death,  and  that  post  mortem  there  was 
widespread  degeneration  of  the  epithelial  cells  of  the  convoluted 
tubules. 

Isocytolysins. — Observations  such  as  these  naturally  raised  the 
question  whether  cytotoxin  production  only  occurs  when  cells  from 
a  given  animal  are  injected  into  an  animal  of  an  alien  species,  i.  e., 
whether  heterocytotoxin  (sive  heterolysiri)  formation  only  is  possible, 
or  whether  something  analogous  does  not  occur  when  cells  from  one 
animal  are  injected  into  another  one  of  the  same  species.  Ehrlich 
and  Morgenroth  could  show  that  the  production  of  such  isocytotoxins 
(sive  isolysins)  can  actually  occur,  for  on  injecting  goats  with  goat 
blood  he  found  that  the  serum  of  the  injected  animals  had  become 
hemolytic  for  goat  corpuscles.  Metschnikoff  similarly  found  that 
an  isospermatoxin  is  formed  when  a  guinea-pig  is  injected  with 
spermatozoa  from  another  guinea-pig,  as  is  evidenced  by  the  fact 
that  such  a  serum  will  rapidly  immobilize  the  spermatozoa.  This 
was  first  shown  in  vitro,  but  subsequently  also  established  for  the 
living  animal,  for  on  injecting  male  mice  with  a  corresponding 
serum  they  were  rendered  sterile  and  coincidently  the  remarkable 
observation  was  made  that  the  semen  of  such  animals  had  lost  its 
antigenetic  properties,  viz.,  that  upon  injection  into  other  animals 
it  had  lost  the  power  of  calling  forth  a  corresponding  formation  of 
spermatotoxin . 

Auto-antibodies. — Further  observation  along  these  lines  then  led 
to  the  important  discovery  that  iso-antibody  formation  not  only 
is  possible,  but  that  auto-antibodies,  viz.,  antibodies  against  the 
cells  of  the  sauie  ariimal  which  ,fur,nis,heol  the  cells,  can  also  be 


IMMUNE  OPSONINS  AND  BACTERIOTROPINS  101 

produced.  The  demonstration  of  this  possibility  is,  of  course, 
most  important  from  the  standpoint  of  animal  pathology,  for  it 
raises  the  question  whether  some  of  the  symptoms  occurring  in 
diseases  in  which  active  cellular  destruction  is  known  to  occur,  or 
even  some  of  the  pathological  lesions,  may  not  be  the  outcome  of  the 
formation  of  autocytotoxins.  Observations  in  this  direction  are  as 
yet  too  meager  to  warrant  any  far-reaching  conclusions.  I  would 
merely  call  to  mind  the  now  well-established  fact  that  the  serum  in 
various  pathological  conditions  has  been  shown  to  be  hemolytic  for 
the  red  cells  of  other  individuals,  and  while  there  is  evidence  to  show 
that  the  cells  of  the  same  individual  (i.  e.,  the  one  furnishing  the 
serum)  are  more  resistant,  the  thought  naturally  arises,  whether 
the  anemia  which  is  so  frequent  in  the  very  diseases,  in  which  auto- 
hemolysins  have  been  demonstrated,  viz.,  syphilis,  tuberculosis, 
and  cancer,  may  not  in  a  measure  be  due  to  the  action  of  such 
antibodies. 

It  has  been  argued  that  if  such  antibody  formation  actually  did 
take  place  the  harm  done  would  be  progressive,  unless  indeed  an  anti- 
autocytotoxin  formation  in  turn  were  to  occur.  The  production 
of  such  bodies  also  has  actually  been  demonstrated  by  a  number 
of  observers  (Bordet,  Miiller,  Ehrlich,  and  Morgenroth),  and  it  has 
been  shown,  moreover,  that  these  substances  are  of  the  nature  of 
anti-amboceptors.  Of  their  role  in  the  animal  organism,  however,  as 
well  as  that  of  the  auto-antibodies  themselves,  our  knowledge  is  still 
most  imperfect  and  a  discussion  of  the  various  possibilities  would  at 
present  amount  to  little  more  than  a  philosophical  discourse. 

Immune  Opsonins  and  Bacteriotropins. — Further  studies  have  shown 
that  in  addition  to  the  various  types  of  antibodies  which  we  have 
considered  so  far  there  are  still  others. 

We  have  had  occasion  to  point  out  in  a  previous  chapter  that  in 
the  course  of  various  infections,  substances  appear  in  the  blood  which 
prepare  the  corresponding  organisms  for  phagocytosis,  but  which 
differ  from  the  normal  opsonins  in  their  greater  thermostability; 
these  bodies  have  been  designated  as  immune  opsonins  or  bacterio- 
tropins.  Bodies  of  this  character  have  been  demonstrated  following 
immunization  with  the  streptococcus,  pneumococcus,  staphylococcus, 
meningococcus,  the  cholera  vibrio,  the  typhoid  and  paratyphoid 
bacillus,  the  dysentery  bacillus,  the  tubercle  bacillus,  the  plague 
bacillus,  and  the  anthrax  bacillus.  Of  their  mode  of  action  nothing 


102  ANTIGENS  AND  ANTIBODIES 

definite  is  known.  Neufeld  inclines  to  the  belief  that  they  produce 
some  alteration  in  the  physicochemical  status  of  the  cell,  in  virtue 
of  which  some  constituent  of  the  cell  body  is  transformed  into  a 
soluble  modification  which  now  surrounds  the  organism  with  a 
delicate  layer  and  serves  to  attract  the  leukocytes.  As  I  have  already 
pointed  out,  the  tropins  can  act  independently  of  the  presence  of 
complement  and  are  thus  of  simpler  structure  than  the  opsonins 
proper — both  those  occurring  in  normal,  as  well  as  those  found  in 
immune  serum.  The  quantity  of  these  substances  which  may  occur 
in  immune  sera  is  sometimes  very  considerable  and  much  greater 
than  that  found  in  normal  blood;  for  whereas  the  latter  can  scarcely 
be  diluted  more  than  fifty  times  before  it  loses  its  specific  action, 
immune  serum  may  at  times  still  call  forth  phagocytosis  when 
diluted  a  thousandfold. 

Antiferments. — Another  group  of  antibodies  are  the  so-called  anti- 
ferments  which  are  specifically  directed  against  the  corresponding 
ferments.  Substances  of  this  order  have  been  observed  in  the  blood 
serum  after  immunization  with  rennin,  pepsin,  trypsin,  tyrosinase, 
thrombin,  urease,  lactase,  lipase,  laccase,  etc.,  the  corresponding 
antiferments  being  accordingly  designated  as  antitrypsin,  antipepsin 
antirennin,  etc. 

The  discovery  of  bodies  of  this  order  is  especially  interesting  as 
it  throws  some  light  on  the  vexed  question:  Why  do  the  various 
cells  of  the  body  not  digest  themselves?  In  former  years  when  the 
digestive  ferments  of  the  stomach  and  pancreas  were  the  only  ones 
known  to  occur  in  the  animal  body,  it  was  a  source  of  wonder  why 
the  corresponding  digestive  juices  did  not  digest  the  organs  in  which 
they  were  produced.  At  the  present  time  when  we  know  that  pro- 
teolytic  ferments  are  present  in  every  cell  we  may  well  wonder  why 
the  whole  body  does  not  digest  itself.  If  leukocytes  are  removed 
from  the  body  and  suspended  in  saline  or  Ringer's  fluid,  self-digestion 
begins  after  a  relatively  short  time  and  leads  to  the  complete  destruc- 
tion of  the  cells,  even  though  the  access  of  bacteria  be  prevented. 
If,  on  the  other  hand,  the  cells  are  suspended  in  normal  serum, 
autodigestion  does  not  occur,  and  if  such  serum  be  tested  against 
a  solution  of  trypsin  it  can  be  shown  that  it  possesses  marked  anti- 
tryptic  properties.  The  discovery,  then,  that  the  antitryptic  con- 
tent of  the  blood  can  be  materially  increased  by  immunization 
with  trypsin,  suggests  the  possibility,  at  least,  that,  even  normally, 


ALB  UMINOL  YSlNS  103 

antiferment  production  occurs  in  the  animal  body  and  renders  the 
conclusion  not  unwarrantable  that  these  antiferments  are  essential 
to  the  life  of  the  whole  organism. 

Antilipoids. — Still  more  recent  studies  have  brought  to  light  another 
interesting  class  of  antibodies  which  are  not  "tuned"  to  any  par- 
ticular cell,  so  far  as  our  present  knowledge  goes,  but  which  have  the 
power  of  reacting  with  substances  belonging  to  the  group  of  lipoids. 
Such  antibodies  have  been  discovered  in  patients  suffering  from 
syphilis,  yaws,  frambesia,  trypanosomiasis,  cancer,  leprosy,  etc.,  and 
may,  for  the  present,  be  spoken  of  as  antilipoids.  Their  reaction 
with  the  corresponding  lipoids  is  characterized  by  the  fact  that  if 
complement  be  present  at  the  time  this  is  fixed  to  a  greater  or  less 
degree,  so  that  upon  the  subsequent  addition  to  the  mixture  of 
lipoid,  antilipoid,  and  complement,  of  washed  red  corpuscles  and  a 
suitable  hemolytic  amboceptor,  hemolysis  either  does  not  occur  at 
all,  or  is  more  or  less  impeded.  This  remarkable  phenomenon  is 
the  basis  of  the  so-called  Wassermann  test  for  syphilis,  and  as  such 
constitutes  one  of  the  most  important  discoveries  of  medicine  (see 
Wassermann  reaction). 

Albuminolysins. — I  have  pointed  out  above  that  upon  injecting 
albumins,  either  of  vegetable  or  animal  origin,  into  an  animal  of  an 
alien  species,  antibodies  are  formed  which  are  known  as  precipitins, 
and  are  characterized  by  their  ability  to  form  a  precipitate,  when 
brought  together  with  the  corresponding  antigenic  substances. 
The  allergic  state  which  develops  as  the  result  of  the  injection  of 
foreign  albumins  can  manifest  itself  in  still  other  ways,  however, 
If  a  guinea-pig  is  thus  injected  with  normal  horse  serum,  for  example, 
and  an  interval  of  from  ten  to  thirteen  days  is  allowed  to  elapse,  it 
will  be  noted  that  the  second  injection  is  followed  by  most  alarm- 
ing symptoms — intense  dyspnea  and  marked  drop  of  tempera- 
ture ( Theobald  Smith  phenomenon) — which  frequently  end  in  death. 
Corresponding  symptoms  occur  in  other  animals  and  may  follow 
the  introduction  of  almost  any  foreign  albumin  by  parenteral  chan- 
nels. Evidently  the  first  injection  sensitizes  the  animal  to  subse- 
quent injections  and  the  thought  naturally  suggested  itself  that  here 
also  antibodies  may  play  a  role.  What  these  antibodies  are  is  still 
a  matter  of  surmise.  Some  investigators  have  attempted  to  identify 
them  with  the  precipitins,  while  others  look  upon  them  as  a  peculiar 
type  of  lysins,  which  we  may  accordingly  term  albuminolysins, 


104  ANTIGENS  AND  ANTIBODIES 

and  suppose  that  during  the  interaction  between  the  lysin  and  its 
antigen,  at  the  time  of  the  second  injection,  highly  poisonous 
intermediary  products  are  formed  to  which  the  peculiar  symptoms 
are  in  turn  due. 

Anaphylaxis. — As  the  injected  animal  has  evidently  become  more 
sensitive  to  the  action  of  the  foreign  albumin  than  it  was  before  the 
first  injection,  which  usually  does  not  give  rise  to  any  serious  symp- 
toms, Richet  suggested  the  term  anaphylaxis  to  express  this  condition 
of  hypersusceptibility,  in  contradistinction  to  prophylaxis,  diminished 
susceptibility  or  immunity,  in  the  older  sense  of  the  word.  This 
term  has  now  been  generally  accepted,  and  the  more  or  less  threaten- 
ing symptoms  which  follow  the  second  injection  are  accordingly 
spoken  of  as  the  anaphylactic  shock.  French  writers,  more  particu- 
larly, refer  these  symptoms  to  a  special  anaphylactic  reaction  product 
which  they  term  anaphylactin.  v.  Pirquet,  as  we  have  already  seen, 
has  introduced  the  non-committed  term  allergia  to  denote  the  changed 
mode  of  reaction  on  the  part  of  the  injected  animal  (no  matter  what 
antigen  has  been  used)  and  speaks  of  the  antigenic  substances  as 
the  allergens  and  the  reaction  products  as  the  corresponding  ergins. 
According  to  his  ideas,  anaphylaxis  is  thus  merely  one  form  in  which 
the  general  allergia  can  express  itself.  In  a  subsequent  chapter  we 
shall  have  occasion  to  deal  with  this  problem  in  greater  detail,  and 
we  hope  to  show  that  the  antibodies  which  are  especially  involved 
in  the  anaphylactic  reaction,  play  an  important  role  in  the  symptom- 
atology of  many  diseases. 

The  brief  survey  of  the  manner  in  which  the  animal  body  responds 
to  the  parenteral  introduction  of  foreign  cells  and  cell  derivatives, 
which  has  just  been  given,  imperfect  and  condensed  though  it  be,  is 
probably  sufficient  to  show  that  a  field  of  work  has  been  opened  up 
which  offers  a  most  alluring  perspective  to  the  investigator,  both  in 
medicine  and  general  biology.  During  the  few  years  that  it  has 
been  tilled,  the  returns  have  already  been  wonderful  in  their  diver- 
sity and  value,  and  we  have  every  reason  to  suppose  that  a  great  deal 
of  the  future  progress  of  medicine  will  lie  in  this  direction.  We  have 
already  a  host  of  experimental  facts  which  only  await  their  proper 
interpretation,  before  they  will  become  important  stepping  stones 
toward  still  more  important  findings.  Among  the  many  able  investi- 
gators who  are  closely  associated  with  progress  along  these  lines,  one 


ANAPHYLAXIS  105 

stands  out  prominently  above  all  others,  because  he  has  furnished 
us  with  a  working  hypothesis  which  satisfactorily  explains  many 
observations  that  have  been  made  in  this  field,  and  because  its  study 
has  opened  up  new  avenues  of  research  along  which  much  fruitful 
work  has  already  been  accomplished.  That  man  is  Paul  Ehrlich, 
and  in  the  subsequent  chapter  we  shall  endeavor  to  show  that  on 
the  basis  of  his  now  world-famous  side-chain  theory  the  formation 
of  the  many  groups  of  antibodies  with  which  we  have  already 
become  acquainted  can  be  explained  and  their  specific  properties 
accounted  for. 


CHAPTER  VIII 
THE  SIDE-CHAIN  THEORY 

WHEN  it  was  discovered  that  the  injection  of  the  serum  of  an 
animal  that  had  been  rendered  immune  to  diphtheria  could  protect 
another  animal  against  infection  with  the  corresponding  organism, 
and  could  even  arrest  the  disease  after  this  had  actually  developed, 
the  question  naturally  arose  how  this  remarkable  effect  could  be 
explained.  Different  possibilities,  of  course,  suggested  themselves. 

Roux  and  Buchner  at  first  expressed  the  opinion  that  the  anti- 
toxin produced  its  effect  by  acting  upon  the  cells  of  the  body  in  such 
a  manner  as  to  increase  their  resistance,  or  to  diminish  their  suscep- 
tibility to  the  corresponding  toxin.  In  other  words,  they  imagined 
that  the  antitoxin  called  forth  a  rapid  immunization  of  the  cell. 
This  view  was  abandoned  when  it  could  be  shown  that  the  addition 
of  antitoxic  serum  to  a  toxin  outside  of  the  body  is  capable  of  pre- 
venting the  effect  of  the  latter,  when  the  mixture  is  subsequently 
injected  into  the  body. 

Ehrlich  first  demonstrated  this  with  the  blood  of  mice  which  had 
been  immunized  against  the  vegetable  toxin  ricin.  He  found  that 
several  multiples  of  the  minimal  fatal  dose  of  this  substance  could 
be  injected  into  animals,  without  producing  any  toxic  symptoms,  if 
an  appropriate  amount  of  blood  from  a  correspondingly  immunized 
animal  (antiriciri)  had  previously  been  added  to  the  ricin  solution. 
Eraser  similarly  found  that  the  antitoxin  directed  against  snake 
poison  (antiveniri)  acted  much  more  energetically,  if  it  was  first 
mixed  with  the  corresponding  toxin  (venin)  outside  of  the  body, 
than  when  its  injection  immediately  followed  or  preceded  that  of 
the  toxin.  Analogous  experiments  with  toxic  eel  serum,  crotin,  and 
the  hemolytic  component  of  the  tetanus  toxin  (tetanolysin)  led  to 
similar  results. 

These  observations  also  rendered  untenable  the  view  that  the  anti- 
toxin only  acquires  active  properties  as  the  result  of  a  ferment-like 
transformation  of  the  substance  on  the  part  of  the  body  cells. 


THE  SIDE-CHAIN  THEORY  107 

Evidently  the  antitoxin  acts  directly  upon  the  toxin,  and  in  inter- 
preting the  findings  in  vitro,  different  possibilities  again  arise.  It  is 
thus  conceivable  that  the  antitixon  may  destroy  the  toxin.  This 
view,  however,  has  also  been  disproved  by  a  number  of  observations. 
Buchner  thus  showed  that  a  mixture  of  tetanus  toxin  and  antitoxin 
which  was  "neutral"  for  mice  was  still  toxic  for  guinea-pigs.  Roux 
and  Calmette  further  ascertained  that  a  mixture  of  snake  venom  and 
antivenin  which  was  non-toxic  for  a  given  animal,  became  toxic  again 
on  heating.  This  would  evidently  be  out  of  the  question  if  the  non- 
toxicity  of  the  mixture  had  been  owing  to  a  destruction  of  the  toxin 
by  the  antitoxin.  Martin  and  Cherry  then  pointed  out  that  this 
result  is  obtained  only  if  not  too  long  an  interval  has  elapsed  after 
bringing  toxin  and  antitoxin  together,  and  that  a  restitution  of  the 
toxic  effect  is  no  longer  possible  if  a  certain  time  limit  has  been 
passed.  They  could  show,  as  a  matter  of  fact,  that  toxin  and  anti- 
toxin do  not  interact  instantaneously,  and  that  at  first  toxin  and 
antitoxin  coexist  in  the  free  state,  the  velocity  of  reaction  depending 
very  largely  upon  the  concentration  of  the  two  solutions  and  the 
temperature. 

In  the  light  of  these  findings  Roux  and  Calmette's  original  observa- 
tions do  not  disprove  the  idea  that  the  antitoxin  destroys  the  toxin. 
That  this  actually  does  not  occur  was,  however,  conclusively  shown 
by  Morgenroth  in  Ehrlich's  laboratory;  for  on  treating  a  mixture  of 
Cobra  toxin  and  antitoxin  that  had  been  kept  for  more  than  a  week, 
i.  e.,  for  a  period  of  time  that  was  more  than  sufficient  completely  to 
destroy  any  toxic  effect  by  the  antitoxin,  with  dilute  acids,  and  on 
then  heating  the  acid  mixture  for  some  time  at  100°  C.  the  original 
quantity  of  toxin  was  again  obtained  in  its  entirety.  A  destruction 
of  the  toxin  by  the  antitoxin  had  thus  been  satisfactorily  disproved. 

Evidence  such  as  this  is,  of  course,  strongly  suggestive  that  the 
inactivation  of  the  toxin  by  the  antitoxin  is  due  to  the  occurrence 
of  a  chemical  interaction  between  the  two,  and  that  the  specific 
effect  of  the  toxin  disappears  because  the  substance  is  chemically 
bound  by  the  antitoxin.  This  view,  which  was  first  expressed  by 
Ehrlich,  is  the  one  now  generally  held  and  forms  the  basis  of  our 
modern  conception  of  the  production  of  antibodies  and  their  specific 
effect. 

Further  studies  have  shown,  as  a  matter  of  fact,  that  the  same 
principle  applies  to  the  interaction  between  other  antigens  and  their 


108  THE  SIDE-CHAIN  THEORY 

antibodies.  If  washed  red  corpuscles  are  thus  brought  together  with 
a  corresponding  hemolytic  amboceptor  and  are  incubated  at  body 
temperature,  the  amboceptor  is  anchored  to  the  corpuscles  and  can 
no  longer  be  removed  by  washing.  That  an  interaction  has  actually 
taken  place  may  be  shown  by  adding  fresh  complement,  when  hemo- 
lysis  will  promptly  occur.  Analogous  results  are  obtained  with  the 
bacteriolytic  amboceptors,  agglutinins,  and  precipitins,  and  as  in  the 
case  of  the  antitoxins  it  may  here  also  be  shown  that  no  destruction 
of  the  antigen  takes  place.  If  milk  and  a  corresponding  precipitin 
(lactoserum)  are  thus  brought  together,  a  precipitate  of  antibody 
casein  is  formed,  and  if  this  is  boiled,  after  careful  washing  in  normal 
salt  solution,  the  precipitate  dissolves,  and  in  the  resulting  solution 
unchanged  casein  can  be  demonstrated,  which  may  in  turn  be 
precipitated  by  the  addition  of  a  new  portion  of  antiserum.  The 
precipitin  can  similarly  be  recovered  by  treating  the  precipitate 
with  T"Q-  sodium  hydrate  or  sulphuric  acid. 

While  a  destructive  action  on  the  part  of  the  antibody  upon  the 
antigen  can  thus  be  excluded,  the  latter  may  be  destroyed  secondarily 
in  consequence  of  the  activity  of  some  additional  factor.  This 
actually  takes  place  when  complement  acts  upon  cells  that  have 
been  brought  together  with  corresponding  cytotoxic  (lytic)  ambo- 
ceptors. The  term  lysin,  of  course,  suggests  that  it  is  the  amboceptor 
that  is  lytic,  but  it  should  not  be  forgotten  that  the  lytic  effect  only 
occurs  when  complement  is  present,  that  the  latter  really  is  the  lytic 
agent,  and  that  the  amboceptor  itself  produces  no  appreciable  effect 
upon  the  antigenic  cells. 

Chemical  Nature  of  Antigen-antibody  Interaction. — The  assump- 
tion that  the  interaction  between  antigens  and  antibodies  is  of 
a  chemical  nature  carries  with  it  the  inference  that  the  reacting 
substances  must  combine  with  one  another  in  certain  definite 
proportions  or  multiples  thereof,  viz.,  that  if  one  unit  of  anti- 
toxin will  neutralize  one  unit  of  toxin,  ten  units  of  the  one  should 
combine  with  ten  of  the  other,  twenty  with  twenty,  etc.  The  inves- 
tigation of  this  particular  side  of  the  problem  has  led  to  a  great  deal 
of  controversy  arising  from  erroneous  interpretations  of  various  obser- 
vations, owing  to  imperfections  in  technique,  lack  of  knowledge  of 
detail,  etc.,  and  has  consequently  been  productive  of  an  enormous 
amount  of  labor,  for  much  of  which  we  are  indebted  to  Ehrlich  and 
his  school. 


CHEMICAL  NATURE  OF  ANTIGEN-ANTIBODY  INTERACTION  109 

From  the  very  nature  of  things  the  interrelation  between  toxins 
and  antitoxins  has  received  the  greatest  amount  of  attention,  for 
here  we  are  dealing  with  substances  which  react  in  solution  and  whose 
behavior  is  thus  more  readily  open  to  investigation  and  interpreta- 
tion than  in  the  case  of  those  phenomena  which  occur  between 
highly  complex  components,  such  as  animal  and  vegetable  cells  and 
their  antibodies.  It  may  be  said  in  advance,  however,  that  notwith- 
standing many  observations  which  at  first  tended  to  suggest  that 
the  character  of  the  interaction  between  toxins  and  antitoxins  was 
of  a  different  nature  than  that  between  the  other  antibodies  and 
their  antigens,  more  detailed  investigations  have  shown  that  this 
difference  is  more  in  appearance  than  in  fact. 

A  number  of  modern  investigators  have  attempted  to  explain 
the  laws  which  have  been  found  to  govern  the  action  of  antibodies 
upon  their  antigens  upon  a  purely  physical  basis,  but,  although 
many  observations  may  be  interpreted  as  supporting  their  view,  one 
factor  is  not  explained  upon  these  grounds,  and  that  is  the  remarkable 
specificity  of  the  antibodies.  Erhlich's  side-chain  theory,  on  the 
other  hand,  which  rests  upon  a  purely  chemical  interpretation  of  the 
phenomena  of  antigen  antibody  interaction,  satisfactorily  accounts 
for  this,  so  that,  even  though  we  admit  the  validity  of  the  physical 
theory  in  the  explanation  of  certain  phenomena  we  must  still  adhere 
to  the  chemical  side.  All  the  facts  which  have  been  observed  when 
toxins  and  antitoxins  interact  can  certainly  be  explained  upon 
chemical  grounds.  In  the  case  of  the  other  antigens  and  their 
antibodies,  we  may  admit  that  physical  processes  may  play  a  role, 
but  in  addition  to  these,  chemical  action  unquestionably  also  takes 
place.  A  somewhat  more  detailed  account  of  certain  studies  along 
these  lines  will  serve  to  bring  out  some  of  the  difficulties  which  have 
been  encountered. 

As  I  have  pointed  out,  if  we  conceive  that  toxin  and  antitoxin 
unite  with  one  another  chemically,  then  we  would  expect  that 
definite  quantities  of  the  one  or  multiples  thereof  would  unite 
with  corresponding  quantities  or  multiples  of  the  other.  To  use  a 
common  example — if  40  parts  by  weight  of  sodium  hydrate  unite 
with  36.5  parts  by  weight  of  hydrochloric  acid,  according  to  the 
equation : 

NaOH        +      HC1       =      NaCl      +      H2O 

m.  weight,  40         m.  w.,  36.5 


110  THE  SIDE-CHAIN  THEORY 

then  2  X  40  parts  of  NaOH  will  unite  with  2  X  36.5  parts  of  HC1, 
and  3  X  40  NaOH  with  3  X  36.5  HC1,  etc. 

When  this  point  was  first  investigated  with  toxin-antitoxin  mix- 
tures it  was  found  that  starting  with  an  apparently  neutral  mixture 
the  injection  of  several  multiples  of  this  proved  highly  toxic;  in  other 
words,  whereas  the  original  mixture  was  apparently  perfectly  innoc- 
uous, a  fatal  result  was  obtained  if  from  two  to  five  times  as  much 
toxin'  was  used,  and  this  treated  with  corresponding  multiples  of 
antitoxin.  Upon  first  consideration  such  a  result  would  seem  entirely 
contradictory  to  the  idea  that  the  toxin  and  antitoxin  neutralize  one 
another  in  a  chemical  sense.  Further  investigation,  however,  has 
shown  that  the  contradiction  is  only  apparent,  and  that  the  law  of 
multiples  does  hold  good  for  the  toxin-antitoxin  interaction,  but 
that  it  is  absolutely  essential  in  such  experiments  to  neutralize  the 
original  mixture  with  such  exactness  that  not  even  a  minimal 
fraction  of  toxin  is  present  in  excess  of  the  antitoxin.  If  this  is  not 
the  case,  one  can  readily  conceive  that  even  though  the  original 
mixture  were  non-fatal,  several  multiples  thereof  might  very  readily 
be  so.  It  is  hence  imperative  that  the  original  mixture  should  be  so 
standardized  that  its  injection  does  not  cause  the  slightest  symptom 
of  disease.  If  this  is  carefully  done  then  it  will  be  found  that  the 
law  of  multiples  actually  does  hold  good,  and  this  law,  as  a 
matter  of  fact,  forms  the  basis  of  Behring  and  Ehrlich's  method  of 
standardizing  the  diphtheria  antitoxin  of  the  market,  in  which  the 
unavoidable  source  of  error  does  not  amount  to  more  than  from 
0.1  to  1  per  cent,  (see  Preparation  of  Diphtheria  Antitoxin). 

If,  now,  we  come  to  apply  the  law  of  multiples  to  the  study  of  the 
other  antibodies,  we  find  that  the  possible  sources  of  error  in  the 
concrete  interpretation  of  the  actual  findings  are  still  greater,  and 
it  may  not  be  out  of  place  to  refer  in  some  detail  to  some  of  the 
difficulties  which  have  been  here  encountered  and  the  manner  in 
which  they  have  been  met.  We  may  say  in  advance,  however,  that 
no  observations  have  been  made  which  would  tend  to  exclude  the 
interaction  between  these  antigens  and  their  antibodies  from  the 
law  of  multiples,  as  it  has  been  established  for  toxin-antitoxin 
mixtures. 

Starting  with  1  c.c.  of  an  emulsion  of  an  agar  slant  culture  of  a 
given  organism  in  15  c.c.  of  normal  salt  solution,  and  treating  this 
with  an  equal  volume  of  an  agglutinating  serum  in  varying  dilutions, 


CHEMICAL  NATURE  OF  ANTIGEN-ANTIBODY  INTERACTION  111 

Eisenberg  and  Volk  term  that  quantity  of  serum  an  agglutinin  unit, 
which  will  bring  about  partial  agglutination  of  the  contained  organ- 
isms in  twenty-four  hours.  If,  then,  constant  quantities  of  the 
bacterial  emulsion  (e.  g.,  1  c.c.)  are  treated  with  an  increasing  number 
of  agglutinin  units  it  will  be  observed  that  the  bacteria  have  the 
power  of  absorbing  an  enormous  excess  of  agglutinin  beyond  the 
amount  that  is  actually  required  to  produce  agglutination.  If, 
moreover,  the  number  of  units  that  is  actually  absorbed  is  compared 
with  the  number  added,  the  interesting  fact  develops  that  with 
increasing  concentration  of  the  agglutinins  the  absolute  absorption 
by  the  bacteria  rises,  while  the  absorption  coefficient,  i.  e.,  the  ratio 
between  the  number  of  units  added  and  the  amount  absorbed, 
falls.  This  is  well  shown  in  the  accompanying  table  which  is 
taken  from  Eisenberg  and  Volk. 

ANTITYPHOID  SERUM,  ZOROASTER  III.      AGGLUTINATION  VALUE  =  45,000  UNITS 

Agglutinin  units     Agglutinin  units     Coefficient  of 

Serum  dilution.  added.  absorbed.  absorption. 

1  to  20000 2  2  1.0 

1  to  2000 22  22  1.0 

Ito  1000 45  45  1.0 

1  to  600 75  75  1.0 

1  to  500 90  89  0.99 

Ito  200 225  210  0.93 

1  to  100 450  400  0.88 

Ito  20  ..'....  2250  1650  0.73 

Ito  4 11250  6750  0.60 

Ito  2 22500  12500  0.56 

Ito  1 45000  22500  0.50 

The  question  then  arises  how  to  explain  the  apparent  paradox 
that  the  same  quantity  of  bacteria  which  can  only  absorb  12,500 
units  out  of  22,500  that  have  been  offered  can  actually  absorb 
22,500  when  brought  in  contact  with  a  proportionately  larger  amount. 
Upon  first  consideration  the  thought  of  a  chemical  union  between 
agglutinin  and  agglutinable  substance  would  seem  to  be  out  of  the 
question.  Various  explanations,  however,  have  been  offered,  any 
one  of  which  would  show  that  the  paradox  is  in  reality  only  apparent. 
As  will  be  seen  later  on  there  are  reasons  for  supposing  that  an 
agglutinating  serum  may  contain  not  only  one  single  agglutinin,  but 
a  number  of  agglutinins  which  correspond  to  the  presence  of  an 
equal  number  of  agglutinable  substances  (agglutinogens)  in  the  body 


112  THE  SIDE-CHAIN  THEORY 

of  the  bacillus;  as  experience,  moreover,  has  shown  that  different 
antigens,  even  though  closely  related,  may  differ  very  considerably 
in  their  antibody  forming  power,  we  may  assume  that  the  number 
of  agglutinable  molecules  in  a  unit  of  bacterial  emulsion  is  different 
from  that  of  the  various  agglutinins  in  a  unit  of  the  corresponding 
serum. 

Supposing,  now,  that  in  the  former  there  were  present  100  mole- 
cules of  the  agglutinable  substance  a,  50  of  the  agglutinable  sub- 
stance b,  and  20  of  c,  while  in  the  antiserum  there  were  present  for 
each  unit  100  molecules  of  aerglutinin  A,  20  of  B,  and  2  of  C,  the 
100  a's  would  then  unite  with  the  100  A's,  20  6's  with  the  20  £'s, 
and  the  2  c's  with  the  2  C's.  There  would  then  be  remaining  30 
unsatisfied  molecules  of  6  and  18  of  c.  If,  therefore,  a  second  unit 
of  agglutinating  serum  were  now  added,  20  of  the  remaining  6's 
would  take  up  the  20  newly  added  B's  and  2  of  the  remaining  18 
c's  the  2  new  portions  of  C.  There  would  now  remain  10  molecules 
of  b  and  16  of  c,  while  the  100  ^4's  from  the  second  agglutinating 
unit  would  be  left  over.  This  would  represent  exactly  what  we  see 
in  the  table  above,  viz.,  that  even  though  agglutinins  in  excess  be 
present  the  bacterial  emulsion  can  still  take  up  more  agglutinin  if 
more  is  added. 

The  reason  for  this  apparent  paradox  is  now,  of  course,  self- 
evident,  and  lies  in  the  fact  that  we  have  been  mentally  in  the  habit 
of  ascribing  the  agglutinating  properties  of  a  given  serum  to  a 
single  substance;  whereas  there  is  good  evidence  to  show  that  this 
is  not  necessarily  the  case,  that  on  the  contrary  the  agglutinating 
effect  may  be  due  to  a  number  of  so-called  partial  agglutinins,  to 
which  a  similar  number  of  agglutinogens  correspond,  and  that 
the  quantities  present  in  the  serum  do  not  tally  with  those  in  the 
bacterial  emulsion.  The  Eisenberg  phenomenon  is  thus  merely  the 
expression  of  the  coexistence  in  the  mixture  of  free  antigen  on 
the  one  hand  and  free  antibody  on  the  other,  the  antigen  being 
in  excess  merely  because  not  enough  antibody  has  been  added. 

Such  a  coexistence  may,  however,  also  be  explained  in  still  other 
ways,  showing  that  there  is  really  nothing  unusual  in  the  phenomenon. 
According  to  the  Guldberg-Waage  law  of  mass  action  the  quantity 
of  two  chemically  reacting  substances  a  and  b  and  their  product  c 
which  may  be  found  at  any  one  time  in  coexistence,  depends  upon 
a  certain  constant  k,  which  varies  only  with  the  nature  of  the 


CHEMICAL  NATURE  OF  ANTIGEN-ANTIBODY  INTERACTION  113 

reacting  substances  and  the  temperature.      Chemical  equilibrium 
will  result  in  accordance  with  the  equation — 

(Ca)n 


in  which  a  and  b  represent  the  reacting  substances,  c  their  product, 
Cc,  the  concentration,  and  n,  m,  and  o  the  respective  number  of 
molecules.  If  we  conceive  that  only  one  molecule  of  the  reacting 
substance  enters  into  play  the  equation  may  be  simplified  so  as  to 
read — 

Ca  .  Cb  = 

As  k  is  constant  it  will  be  seen  that  by  increasing  the  concentra- 
tion of  either  a  or  b  the  concentration  of  the  product  also  must  be 
increased.  If,  now,  we  conceive  k  to  be  infinitesimally  small  or  even 
equal  to  zero,  then  the  concentration  of  either  a  or  6  or  of  both  must 
be  zero,  since  c  itself  cannot  be  zero.  In  this  case  we  would  come  to 
an  end  reaction  where  both  a  and  b  would  be  completely  used  up 
and  only  the  product  remain.  If,  on  the  other  hand,  k  is  infinitely 
large  then  the  concentration  of  c  must  be  correspondingly  small,  and  if 
k  =  GO  then  c  must  equal  0,  which  means  that  no  union  whatever 
occurs  between  a  and  b.  Between  these  two  extremes,  viz.,  k  =  0 
and  k  =  °o,  an  infinite  number  of  variations  is,  of  course,  possible, 
and  it  is  thus  readily  conceivable  that  k  in  the  case  of  the  agglu- 
tinin-agglutinable  substance  reaction  may  be  of  such  a  value  that 
a  partial  reaction  only  is  possible  between  the  two.  This,  of  course, 
is  what  we  see  in  the  Eisenberg  phenomenon,  and  accepting  this 
explanation  we  would  have  additional  proof  that  the  reaction  be- 
tween antigen  and  antibody  is  actually  of  a  chemical  character. 

Still  another  explanation  is  possible  on  the  basis  of  the  so-called 
law  of  distribution.  This  is  based  upon  the  following  considerations : 
If  a  given  substance  a  is  soluble  in  two  solvents  A  and  B  and  if  the 
substance  in  question  is  brought  together  with  A  and  B  simulta- 
neously a  certain  amount  will  be  dissolved  in  A  and  a  certain  amount 
in  B.  The  ratio  between  the  two  amounts  is  a  constant  which  varies 
only  with  the  nature  of  the  substance  in  question  and  the  tempera- 
ture, but  which  is  uninfluenced  by  the  initial  concentration.  From 
a  study  of  the  figures  given  by  Eisenberg  (see  above),  Arrhenius 
8 


114  THE  SIDE-CHAIN  THEORY 

concluded  that  the  peculiar  relationship  between  the  amount  of  agglu- 
tinin  present  in  the  free  state  and  that  taken  up  by  the  bacteria 
could  be  readily  accounted  for  on  the  basis  of  the  law  of  distribution, 
and  he  developed  for  this  particular  case  the  equation 

(Quantity  of  bound  agglutinin)3 

(Quantity  of  free  agglutinin)*   '    =  *  <constaQt) 

The  figures  actually  observed  in  the  experiment  and  those  theo- 
retically expected  are,  indeed,  so  nearly  alike  that  it  would  seem 
unnecessary  to  seek  for  any  further  explanation  of  the  Eisenberg 
phenomenon  (see  accompanying  table).  But  even  though  we 
admit  that  the  manifest  antigen-antibody  reaction  can  thus  be  satis- 
factorily accounted  for  on  the  basis  of  purely  physical  absorption, 
this  in  itself  does  not  preclude  the  possibility  of  a  subsequent 
chemical  interaction  between  the  two  substances. 


No.  of  agglutinin  No.  of  units  absorbed  according      No.  of  units  absorbed  according 

units  added.  to  observation.  to  calculation. 

2  2  1 . 98 

20  20  19.3 

40  40  37.9 

200  180  180.3 

400  340  347.1 

2000  1500  1522.0 

10000  6500  6110.0 

20000  11000  10840.0 


Formation  of  Antibodies. — If  now  we  pass  on  to  a  consideration  of 
the  question  how  the  introduction  into  the  body  of  a  given  antigen 
can  lead  to  the  formation  of  corresponding  antibodies,  we  do  so 
upon  the  basis  that,  even  though  physical  laws  may  be  operative 
during  the  interaction  between  the  two  classes  of  substances,  actual 
chemical  union  must  invariably  occur.  This,  indeed,  constitutes 
the  very  key-stone  of  Ehrlich's  side-chain  theory,  a  study  of  which 
must  now  engage  out  attention.  According  to  Ehrlich's  doctrine, 
we  must  look  upon  every  cell,  stereochemically  speaking,  as  being 
composed  of  a  central  molecular  complex  upon  the  integrity  of 
which  the  life  and  activity  of  the  cell  depends,  and  of  a  variable 
number  of  subsidiary  molecular  groups  which  serve  the  purely  vege- 
tative functions  of  the  cell.  The  former  Ehrlich  appropriately 
designates  as  the  "  Leistungskern,"  or  functional  nucleus  of  the  cell, 
and  the  latter  as  the  so-called  "  Setienketten"  or  side  chains. 


PLATE  I 


Diagrammatic  Representation  of  the  Functional  Nucleus 
of  Three  Different  Types  of  Cells  and  the  Different  Quanti- 
tative Relations  of  the  Various  Side  Chains. 

A.  a=35%,  6=15%,  c  =  10%,  d=15%,  e=15%,/  =  10%,  g  =0,  h=0 

B.  a  =20%,  6=30%,  c=10%,  d=15%.  e  =  15%,/  =  0,  (7  =  10%,  h  =0 

C.  a  =  10%,  6=30%,  c  =  10%,  d  =  15%,  e  =  15%,  /=0,  ^=0,  A=20% 


FORMATION  OF  ANTIBODIES  115 

Through  the  side  chains  the  nutrition  of  the  central  nucleus  may 
be  conceived  to  be  regulated,  and  as  differences  in  function  no  doubt 
presuppose  certain  underlying  differences  in  chemical  composition, 
and  hence  differing  chemical  affinities  for  those  substances  which 
constitute  the  foodstuffs  of  the  cell,  we  may  well  imagine  that  not 
all  the  side  chains  of  a  given  cell  (through  which  the  nutritional 
processes  must  take  place)  are  chemically  alike,  and  that  certain  side 
chains  are  peculiar  to  a  certain  cell  type,  while  others  are  common 
to  all  cells.  This  conception  of  the  chemical  structure  of  the  cell 
may  be  diagrammatically  expressed  by  representing  the  functional 
centre  as  a  sphere,  from  which  variously  colored  rays — the  side 
chains,  emanate,  and  the  difference  between  a  nerve  cell,  for  example, 
and  a  connective-tissue  cell  or  muscle  cell  could  be  expressed  by  the 
presence  of  a  different  percentage  of  rays  of  a  common  color  and  the 
additional  presence  of  special  rays  of  differing  tints.  This  I  have 
attempted  to  illustrate  in  the  accompanying  illustration  (Plate  I). 

It  will  be  seen  that  all  three  types  of  cells  have  a,  6,  c,  d,  and  e 
rays  in  common,  but  that  these  are  present  in  different  percentage 
proportions,  and  that  the  cells  differ  from  one  another  not  only  in 
this  respect,  but  also  in  the  exclusive  presence  of  /  rays  in  the  one, 
of  g  rays  in  the  other,  and  of  h  rays  in  the  third.  Ehrlich  further 
conceives  that  a  given  foodstuff  can  only  be  utilized  by  a  given  cell, 
if  it  possesses  atomic  groups  which  are  capable  of  combining  with 
corresponding  groups  of  the  side  chains.  As  the  latter  are  not  all 
alike  in  their  chemical  structure,  it  is  reasonable  to  suppose  that 
a  definite  relationship  must  exist  between  their  combining  groups 
and  the  combining  groups  of  the  foodstuffs,  such  that  certain 
food  molecules  only  will  be  capable  of  uniting  with  certain  side 
chains. 

To  use  the  frequently  quoted  simile  which  Emil  Fischer  first  applied 
to  the  specific  action  of  ferments,  we  may  say  that  the  combining 
group  of  the  food  molecule  must  fit  a  corresponding  group  of  the 
side  chain  like  a  complex  key  fits  its  special  lock.  To  revert  to  our 
diagram  we  may  express  this  by  assuming  that  only  a  black  food 
molecule  can  combine  with  a  black  side  chain,  only  a  green  one 
with  one  of  its  own  color,  etc. 

All  those  side  chains  which  are  capable  of  combining  with  chemical 
bodies  in  general,  Ehrlich  designates  collectively  as  receptors,  or 
chemoreceptors,  while  those  which  react  with  foodstuffs  more  or  less 


116  THE  SIDE-CHAIN  THEORY 

exclusively,  and  which  accordingly  serve  the  nutrition  of  the  cell, 
are  appropriately  termed  nutriceptors. 

Under  ordinary  conditions  of  cell  life,  we  can  well  imagine  that 
the  cell  receptors  will  have  occasion  to  react  only  with  actual  food- 
stuffs. But  we  can  also  conceive  that  under  abnormal  conditions,  as 
in  the  various  infections,  substances  may  be  brought  to  the  cell  which 
accidentally  possess  an  atomic  group  that  is  identical  in  structure 
with  the  combining  or  haptophoric  group,  as  it  is  termed,  of  the 
usual  food  molecule  to  which  the  special  receptor  is  "tuned." 

To  use  a  homely  simile,  we  may  say  that  while  ordinarily  only  the 
rightful  owner  of  a  house  can  unlock  its  doors,  the  possibility  exists 
that  a  burglar  with  a  master  key  could  similarly  gain  entrance; 
and  to  carry  the  simile  farther;  while  the  entrance  of  the  house- 
owner  would  not  be  attended  by  any  undesirable  consequences,  the 
result  might  be  quite  different  in  the  case  of  the  burglar.  To  return 
to  actual  conditions  we  can  readily  see  that  the  existence  of  such  a 
combining  group  on  the  part  of  a  toxin  molecule,  for  example,  might 
actually  be  fatal  to  the  life  of  the  cell,  supposing,  of  course,  that  the 
Leistungskern  itself  could  be  injured  by  the  toxin.  As  a  matter  of 
fact,  this  is  exactly  what  Ehrlich  supposes  to  occur  in  such  infections 
as  diphtheria,  tetanus,  and  botulismus.  He  conceived  that  the 
toxic  molecule  in  question  must  have  two  distinct  molecular  groups, 
one  of  which — the  haptophoric  group — anchors  the  toxin  to  the  cell 
receptor,  while  the  other — the  toxophoric  group — is  the  actual  bearer 
of  its  toxic  properties. 

If  now  we  imagine  that  the  number  of  toxin  molecules  which  have 
thus  gained  access  to  the  cell,  in  virtue  of  the  identity  in  the  struc- 
ture of  its  haptophoric  group  with  that  of  the  usual  food  molecule 
is  not  sufficiently  large  to  cause  its  destruction,  the  cell  would  never- 
theless suffer  to  a  greater  or  less  extent  owing  to  the  occupation  of 
important  nutriceptors  by  material  that  possesses  no  food  value, 
unless  indeed  it  succeeds  in  freeing  itself  of  its  undesirable  encum- 
brance. When  the  nutriceptors  combine  with  ordinary  food  mole- 
cules we  may  imagine  that  this  union  is  not  permanent,  but  that  the 
food  molecules  are  used  up  chemically  to  supply  the  needs  of  the  cell. 
Coincidently  the  corresponding  receptors  are  again  liberated  and 
placed  in  a  position  where  they  can  combine  with  a  new  set  of  food 
molecules,  and  so  on.  If  the  toxin  molecule,  on  the  other  hand, 


FORMATION  OF  ANTIBODIES  117 

cannot  be  destroyed  in  this  manner  the  cell  must  use  some  other 
method  to  rid  itself  of  the  offending  material. 

According  to  Ehrlich's  concept,  it  accomplishes  this  by  casting  off 
the  receptor  together  with  the  anchored  toxin  molecule.  The  result- 
ing defect  in  its  structure,  the  cell  then  makes  up  by  a  production  of 
new  receptors  of  the  same  kind.  In  accordance  with  Weigert's  law 
of  regeneration  this  new  production,  however,  takes  place  in  excess 
of  the  actual  requirements,  a  condition  of  affairs  which  the  cell 
meets  by  throwing  off  the  unnecessary  number  of  receptors  as  such. 
These  cast-off  receptors  will,  of  course,  have  the  same  combining 
groups  as  the  sessile  ones,  which  had  originally  anchored  the  toxin 
molecule,  and  it  stands  to  reason  that  if  the  toxin  molecule  and  the 
corresponding  free  receptor  are  brought  together  either  within  or 
outside  of  the  body  the  two  will  unite,  the  result  being  indicated  by 
absence  of  toxicity  on  the  part  of  the  mixture.  As  this  is  exactly 
what  happens  when  toxin  and  the  serum  of  a  correspondingly 
immunized  animal  are  brought  together,  Ehrlich  very  properly 
concludes  that  the  antitoxic  properties  of  an  immune  serum  are  due 
to  the  presence  of  free  receptors  which  are  "tuned"  to  the  toxin  in 
question;  in  other  words,  that  the  antitoxin  is  not  newly  formed 
in  the  body,  but  is  identical  with  those  receptors  of  the  cell  which 
render  the  attack  of  the  toxin  upon  the  cell  possible. 

While  this  conception  of  the  nature,  production,  and  mode  of 
action  of  the  antitoxins  originally  had  reference  to  these  only, 
subsequent  observations  led  Ehrlich  to  extend  his  theory  to  the  other 
antibodies  as  well.  But  in  accordance  with  the  facts  observed  it  is 
necessary  to  assume  that  the  structure  of  the  other  antibodies,  viz., 
those  receptors  which  enter  into  relationship  with  such  antigens  as 
the  agglutinable  substance  of  bacteria,  the  precipitable  complex 
of  albumins  and  those  cellular  constituents  which  give  rise  to  lysin 
formation,  must  be  different  from  that  of  the  antitoxic  receptors. 
For  whereas  the  antitoxic  antibodies  merely  combine  with  the  toxins 
to  form  non-toxic  components,  the  other  antibodies  not  only  fix  the 
corresponding  antigens,  but  bring  about  further  changes. 

Ehrlich  very  appropriately  remarks  that  the  mere  fixation  of 
certain  food  molecules  would  not  suffice  to  render  them  available  for 
purposes  of  nutrition,  but  that  with  molecules  of  large  size,  their 
destruction  must  precede  assimilation.  This  could-  be  effected,  if 
the  receptor  in  question  had  not  only  a  haptophoric  group  "tuned" 


118  THE  SIDE-CHAIN  THEORY 

to  the  combining  group  of  the  food  molecule,  but  in  addition,  either 
a  second  group  of  ferment  character  as  part  and  parcel  of  the  same 
receptor,  or  a  second  haptophoric  group  which  might  anchor  ferment 
molecules,  normally  occurring  free  in  the  blood,  when  the  other 
combining  group  is  occupied  by  a  food  molecule  of  a  certain  structure. 

Experimental  investigation  has  shown  that  receptors  of  both  types 
actually  exist,  and  we  may  accordingly  conclude  that  the  antigens 
in  question,  like  the  toxins,  do  not  represent  true  foodstuffs,  but 
become  anchored  to  the  cells  only  because  they  happen  to  possess 
haptophoric  groups  which  are  identical  in  structure  with  those  of 
the  normal  foodstuffs  that  the  particular  receptors  are  in  the  habit 
of  binding.  The  consequence  is  that  here,  also,  the  cell  will  cast 
off  the  useless  receptors  and  produce  the  same  kind  in  unnecessarily 
large  numbers,  the  excess  being  thrown  off  as  in  the  case  of  the  anti- 
toxins. If,  then,  such  free  receptors  meet  with  their  respective  anti- 
gens an  interaction  between  the  two  will  occur  and  this  interaction 
will  manifest  itself  by  agglutination,  precipitation,  lysis,  susceptibility 
on  the  part  of  a  given  cell  to  phagocytosis,  etc.,  as  the  case  may  be. 
We  should  bear  in  mind,  however,  that  the  result,  whatever  it  may 
be,  cannot  be  viewed  as  being  due  to  an  interaction  between  the 
antibody  and  antigen  as  a  whole,  but  as  the  antibody  production 
is,  no  doubt,  the  outcome  of  the  presence  in  the  antigen  of  a  definite 
molecular  complex,  the  visible  effect  is  merely  the  expression  of  an 
interaction  between  the  antibody,  and  that  particular  group;  agglu- 
tination, precipitation,  and  cellular  lysis  are  thus  purely  secondary 
results. 

In  our  illustration  of  the  diversity  of  receptors  which  we  conceive 
to  exist  in  a  given  cell  (Plate  I),  we  have  designated  the  essential 
differences  in  their  general  character  by  differences  in  color,  and  have 
assumed  that  receptors  of  one  color  can  only  combine  with  food 
molecules  of  the  same  color.  The  difference  in  the  structure  of  the 
receptors  is,  however,  best  shown  according  to  Ehrlich's  original 
schema  (see  Plate  II). 

According  to  this  diagram,  then,  we  recognize  three  different  kinds 
of  receptors  or  haptins,  as  they  are  also  called.  Those  of  the  first 
order  possess  only  a  single  combining  or  haptophoric  group,  by  which 
they  unite  with  a  corresponding  group  of  the  respective  antigens. 
The  antitoxins,  antiferments,  tropins,  and  anticomplements  belong 
to' this  category. 


PLATE  II 


Effect  of  immunizing  substance  J.S. 
upon  the  receptors  of  the  order  I,  II 
and  III  (  rl.rll,  rill  )  of  the  cell 

Free  receptors 
(Haptins) 

Effect  of  antibodies 
upon  the  soluble  products 
of  the  cells  or  the  cells 
themselves 

Action  ofantiantibodies 
upon  the  corresponding 
antibodies 

.Toxophoric    , 
„  /Haptophoric  fGr°M 

'W        A, 

> 

^O—  Toxin 

r/-H__f 

f 

%—  Antitoxin 

/  7**     ^ 

N,V%             BodV  Cel1 

jf*       p. 

\3-Agglutinin 

V^V                                VM                1  VnJ 

Haptophoric 

9 

m^^^Insuluble  products     A/^      IJLJ"^ 

I-  Group 

Agglutinin 

4       « 

*M-  Antt- 
agalutinin 

*           i     formation  of 
•v*^*  AgijtutiniHsand            Body  Cell 
o\«>      Precipitins 

^ymophoric 
Group 
Precipitins 
Agglulinins 

Cell  used  for 
immunisation 

^  Cell  used  for   ^ 
immunisation 

•Sf—  Complement 
Jj__Anti- 

used  ^  ^  '^Jf''^t^ 

"C^    complement 

fmmunt   ""•^'.^J^^         ^Complement 

/   W  Amboceptor 

sal  ion         ^^r.^^      (JS^           I  ^A 

^  

/    I       f        A 

^^!^/%^ 

Cytophilic 
m~  Group 

W~ 

H  —  Amboceptor     / 

^       A.        A         / 

Cell  used  for 
immunisation 

-^       X-                                                                             ~v 

^Complement 

^    Cell  used  for      »\ 

WB—  Complement 

£0dj/Ce« 

v*~  ophilic 
Group 

immunisation 

>v        W  —  Amboceptor 

Cytriltjtic  Amboceptors 

^Jn        Anti- 

(Hetnoli/si-ns,  Bacterio- 

(^y    amboceptor 

lysins,  etc  ) 

A       A        A 

Ce/Z  used  for 

immunisation 

Diagrammatic  Representation  of  the  Structure  of  the 
Different  Antibodies  and  their  Relation  to  the  Correspond- 
ing Antigens.  (Taken  from  Asehoff.) 


FORMATION  OF  ANTIBODIES  119 

The  receptors  of  the  second  order  likewise  possess  a  haptophoric 
group  for  the  corresponding  antigen,  and  in  addition  a  special 
ergophoric  group,  as  it  is  called,  by  means  of  which  the  anchored 
antigen  can  be  subjected  to  further  change.  To  this  group  belong 
the  agglutinins  and  the  precipitins. 

It  will  be  noted  that  the  receptors  both  of  the  first  and  the  second 
order  possess  only  a  single  combining  group  with  which  substances 
beyond  the  cell  can  unite.  For  this  reason  they  are  also  spoken  of 
as  uniceptors.  The  receptors  of  the  third  order,  on  the  other  hand, 
possess  two  combining  groups  and  are  hence  termed  amboceptors. 
One  of  these  is  an  ordinary  haptophoric  group  which  anchors  the 
antigen  to  the  cell,  while  the  second  combines  with  the  complement 
of  the  serum  and  is  hence  spoken  of  as  the  complementophilic  group. 
A  special  ergophoric  group  is  not  present,  the  changes  which  occur 
subsequent  to  the  union  between  antigen  and  antibody  being 
effected  by  the  complement  of  the  serum.  To  this  order  belong 
all  the  cy  to  toxins  (or  cytolysins),  the  immune  opsonins,  and  the 
lipoidophilic  antibody  of  Wassermann. 

From  the  above  survey  it  is  quite  evident  that  Ehrlich's  side- 
chain  theory  lends  itself  exceedingly  well  to  experimental  investi- 
gation, and  it  may  not  be  out  of  place  to  consider  in  some  detail 
how  far  the  experimental  facts  support  some  of  the  more  immediate 
conclusions  to  which  the  theory  would  lead. 

We  have  seen  that  according  to  Ehrlich,  the  antibodies  are  not 
formed  de  now,  but  that  they  are  molecular  groups  which  were  once 
part  and  parcel  of  the  cell  upon  which  the  corresponding  antigen  has 
acted.  In  that  case  it  should  be  possible  to  neutralize  the  effect  of 
the  antigen  by  treating  this  with  the  cells  from  which  the  antibody 
is  supposedly  derived,  and  conversely  we  would  expect  that  an 
admixture  of  other  cells  would  not  produce  this  result.  Ransom 
was  one  of  the  first  to  investigate  this  point.  After  poisoning  pigeons 
with  tetanus  toxin  he  found  that  extracts  of  all  the  organs  were  toxic, 
excepting  those  made  from  the  central  nervous  system.  This  would 
suggest  that  the  latter  alone  had  been  able  to  bind  the  toxin.  Wasser- 
mann and  Takaki  then  showed  that  this  is  actually  the  case,  for  on 
rubbing  up  the  same  toxin  with  the  brain  substance  of  guinea-pigs, 
and  injecting  the  mixture  into  animals,  no  deleterious  results  were 
observed,  while  the  liver,  spleen,  adrenal  glands,  muscles,  etc.,  did 
not  have  this  effect.  These  results  are  thus  quite  in  accord  with  what 
we  would  expect  on  the  basis  of  Ehrlich's  theory. 


120  THE  SIDE-CHAIN  THEORY 

By  working  with  animals,  on  the  other  hand,  which  are  either  not 
at  all,  or  but  very  slightly  susceptible  to  the  action  of  the  toxin  in 
question,  such  as  the  turtle,  the  frog,  or  the  alligator,  we  would  expect 
that  the  brain  substance  of  these  would  have  little  if  any  neutral- 
izing action  upon  the  poison.  With  this  supposition  the  facts  are  in 
perfect  accord.  Analogous  results  have  been  obtained  by  Kempner 
with  the  botulismus  toxin,  which  like  the  tetanus  toxin,  possesses 
a  specific  affinity  for  the  nervous  system,  and  we  have  already  seen 
that  it  is  possible  to  remove  hemolytic  and  bacteriolytic  amboceptors 
from  the  respective  sera  by  mixing  these  with  their  corresponding 
antigens. 

The  next  question  which  Ehrlich's  theory  would  suggest  has 
reference  to  the  experimental  basis  for  the  idea  that  the  antibodies 
are  actually  formed  by  the  cells  which  possess  suitable  receptors  for 
the  various  antigens.  Two  possibilities  present  themselves  in  this 
connection.  We  can  conceive,  on  the  one  hand,  that  the  antibodies 
might  be  formed  only  by  those  cells  whose  functional  nucleus  can  be 
deleteriously  influenced  by  the  antigen,  while,  on  the  other  hand,  the 
possibility  exists  that  any  cell  may  produce  antibodies  to  a  given 
antigen,  providing  only  that  it  possesses  a  haptophoric  group  which 
is  capable  of  uniting  with  the  antigen.  Investigations  in  this  direc- 
tion have  led  to  the  conclusion  that  a  mere  union  between  antigen 
and  cell  receptor  is  not  always  sufficient  to  call  forth  antibody  libera- 
tion, but  that  a  special  "Bindungsreiz,"  or  stimulus,  must  also  be 
operative.  Bruck  thus  found  that  on  immunizing  guinea-pigs  with 
two  separate  solutions  of  tetanus  toxin  which  were  several  years  old, 
and  of  which  one  was  still  slightly  toxic,  while  the  other  had  lost  its 
toxicity  altogether,  antitoxin  production  could  only  be  elicited  with 
the  first.  With  the  non-toxic  specimen  this  was  impossible,  even 
though  it  could  be  shown  that  this  still  had  the  power  of  binding 
antitoxin,  which  means,  of  course,  that  its  haptophoric  group  was 
still  intact. 

Bruck  then  argued  as  follows:  If  the  nerve-cell  receptors  of  the 
animal  that  has  been  treated  with  the  non-toxic  product,  i.  e.,  with 
so-called  toxoid,  actually  combine  with  this,  then  the  subsequent 
injection  of  a  dose  of  active  toxin  of  such  amount  as  would  be  just 
sufficient  to  cause  death  in  a  normal  control,  should  now  prove  non- 
fatal.  As  a  matter  of  fact  this  is  exactly  what  occurs  if  the  injection 
of  the  toxin  follows  that  of  the  toxoid  immediately.  If,  on  the  other 


FORMATION  OF  ANTIBODIES  121 

hand,  an  interval  of  twenty-four  hours  is  allowed  to  elapse  between 
the  first  and  the  second  injection  the  latter  will  prove  fatal,  and  it  1 
may  further  be  shown  that  after  such  an  interval  a  dose  which  would 
be  subfatal  for  the  control  is  now  fatal  for  the  toxoid  animal. 

The  interpretation,  of  course,  is  that  the  toxoid  which  was  first 
injected  has  not  only  combined  with  the  nerve-cell  receptors,  but 
has  actually  called  forth  an  increased  production  of  new  receptors  of 
the  same  order,  so  that  there  is  now  present  in  the  nervous  system 
a  larger  number  with  which  the  toxin  molecules  can  combine.  The 
same  experiment  also  shows  that  even  though  the  toxoid  has  called 
forth  such  an  increased  production  of  receptors  this  was  not  followed 
by  their  liberation;  for,  if  this  had  occurred,  the  toxin  molecules 
would  have  met  these  in  the  circulation,  union  would  have  taken 
place  there  .and  the  animal  would  have  remained  alive.  We  are  thus 
forced  to  the  conclusion  that  within  certain  limitations,  antigens,  of 
the  toxin  type  at  least,  can  call  forth  antibody  liberation  providing 
that  the  toxophoric  group  has  not  been  entirely  destroyed. 

As  regards  the  question  whether  antibody  production  can  be 
effected  only  in  those  cells  upon  which  the  toxophoric  group  can 
exert  a  deleterious  effect,  or  whether  the  same  result  can  be  reached 
with  other  cells,  it  is  clear  from  the  experiment  cited  above,  to  show 
that  the  toxins  are  actually  bound  by  those  cells  which  are  susceptible 
to  their  toxic  influence,  that  the  antitoxins  are  also  formed  by  these 
cells.  This  is  further  supported  by  an  experiment  of  Roemer.  This 
investigator  rapidly  immunized  the  right  conjunctiva  of  a  rabbit 
with  abrin  and  then  killed  the  animal.  If  now  the  right  conjunctiva 
was  triturated  with  a  single  fatal  dose  of  abrin  and  the  mixture 
was  injected  into  an  animal,  no  deleterious  result  followed.  If, 
however,  the  same  was  done  with  the  left  conjunctiva  the  animal 
died.  Roemer  accordingly  concludes  that  in  the  right  conjunctiva 
locally  formed  antitoxin  must  have  been  present. 

While  the  evidence  is  thus  quite  conclusive  that  cells  which  are 
susceptible  to  the  toxic  action  of  the  toxin  molecule  may  also  produce 
antitoxins,  there  are  other  facts  to  show  that  this  can  also  occur  in 
non-sensitive  cells.  If,  however,  by  any  chance  the  sensitive  cells 
are  the  only  ones  which  possess  the  necessary  combining  group  for 
the  toxin,  they  will  of  necessity  be  the  only  ones  from  which  anti- 
toxin formation  can  proceed.  We  have  seen  already  that  in  the 
case  of  the  guinea-pig  the  nerve  tissue  is  the  only  tissue  which  can 


122  THE  SIDE-CHAIN  THEORY 

exercise  a  detoxifying  action.  In  the  case  of  the  rabbit,  on  the  other 
hand,  such  an  effect  can  be  produced  not  only  by  the  cells  of  the 
nervous  system,  but  also  by  the  liver  and  the  spleen.  If,  moreover, 
a  guinea-pig  is  injected  with  tetanus  toxin  the  fatal  dose  is  the 
same,  no  matter  whether  the  poison  is  injected  intracerebrally, 
intravenously,  or  subcutaneously.  In  the  rabbit  the  result  is  dif- 
ferent. Intracerebral  injection  of  tiny  doses  readily  leads  to  fatal 
tetanus,  while  much  larger  amounts  can  be  administered  sub- 
cutaneously without  causing  a  fatal  result,  particularly  if  the  larger 
nerve  trunks  in  the  district  in  which  the  injection  is  made  are 
previously  cut.  As  abundant  antitoxin  formation  then  takes  place 
notwithstanding  the  fact  that  the  access  of  the  toxin  to  the  brain 
has  been  excluded  so  far  as  possible,  the  inference,  of  course,  is 
perfectly  warrantable  that  the  antitoxin  in  question  is  largely 
produced  by  cells  which  are  not  susceptible  to  the  toxic  action 
of  the  poison. 

The  same  point  is  also  well  illustrated  in  the  case  of  the  alligator. 
This  animal  is  not  at  all  susceptible  to  the  action  of  tetanus  poison. 
But  notwithstanding  this  fact  the  toxin  rapidly  disappears  from  its 
blood  after  injection,  and  in  its  place  large  amounts  of  antitoxin 
appear.  Were  the  toxin  only  physically  stored  away  in  the  tissues, 
but  not  chemically  bound,  then  we  should  not  expect  antitoxin  for- 
mation, and  the  toxin  should  still  be  demonstrable  in  the  tissues. 
This  is  what  actually  happens  in  scorpions.  Metschnikoff  injected 
such  animals  with  a  thousandfold  quantity  of  the  toxin  as  compared 
with  that  which  is  necessary  to  kill  mice.  The  animals  in  this  case 
were  likewise  not  rendered  ill  and  the  toxin  here  also  disappeared  from 
the  blood.  But  on  testing  for  the  presence  of  antitoxin  none  could 
be  found,  and  even  after  months  it  could  be  shown  that  unchanged 
toxin  was  present  in  the  liver.  The  interpretation  of  these  findings 
upon  the  basis  of  Ehrlich's  side-chain  theory  is  very  simple.  Neither 
the  alligator  nor  the  scorpion  are  rendered  ill  by  the  toxin,  because 
neither  animal  possesses  cells  that  could  be  deleteriously  influenced 
by  the  toxin.  The  alligator,  however,  produces  antitoxin,  because 
its  cells  are  nevertheless  able  to  enter  into  chemical  union  with 
the  toxin,  and  it  is  for  this  reason  that  the  toxin  disappears  from 
the  circulation.  The  scorpion,  on  the  other  hand,  has  no  cells  at  its 
disposal  which  could  unite  chemically  with  the  toxin  and  it  can  hence 
produce  no  antitoxin.  The  poison  here  simply  disappears  because 


FORMATION  OF  ANTIBODIES  123 

it  is  physically  absorbed,  and  it  still  remains  active  for  this  very 
reason  and  because  it  is  not  chemically  bound. 

A  further  consequence  of  Ehrlich's  side-chain  theory  would  be  the 
inference  that  it  should  not  be  possible  to  call  forth  antibody  produc- 
tion by  immunizing  with  exactly  neutralized  antigen-antibody  mix- 
tures, for  in  such  instances  the  haptophoric  group  of  the  antigen  is 
supposedly  in  combination  with  the  corresponding  group  of  the  anti- 
body, and  it  should  hence  not  be  able  to  combine  with  any  receptors 
in  the  body  of  the  injected  animal.  Here  also  the  facts  are  in  accord 
with  the  demands  of  the  theory.  It  is  thus  impossible  to  call  forth 
any  antitoxin  formation  with  accurately  neutralized  toxin-antitoxin 
mixtures.  It  should  be  added,  however,  that  in  such  experiments  it 
is  absolutely  essential  to  have  no  free  toxon  present  beside  the  toxin. 

Toxons  are  poisonous  bodies  which  may  be  present  in  the  diphtheria 
bacillus  cultures  together  with  the  toxins,  but  they  differ  from  these 
in  being  less  toxic.  Like  the  toxins,  however,  they  possess  a  hapto- 
phoric group  which  is  capable  of  combining  with  the  true  antitoxin 
though  the  affinity  of  the  toxon  for  the  antitoxin  is  feebler  than  that 
of  the  toxin.  Since  the  toxon  effect  is  not  acute,  but  only  develops 
after  a  period  of  two  weeks  or  longer,  it  is  clear  that  apparent  neu- 
tralization of  a  toxic  bouillon,  as  tested  by  the  non-development  of 
acute  symptoms,  does  not  imply  the  absence  of  toxons;  and  as  the 
latter  contain  the  same  haptophoric  group  as  the  toxins  it  is  clear 
that  a  mixture  of  both,  which  is  neutralized  by  antitoxin  only  so 
far  as  the  toxins  go,  can  still  call  forth  antitoxin  production.  If, 
on  the  other  hand,  both  toxins  and  toxons  are  neutralized,  then,  as 
I  have  pointed  out,  no  antibody  formation  will  take  place. 

Analogous  experiments  with  cellular  antigens  and  their  corre- 
sponding antibodies  have  led  to  corresponding  results,  though  these 
are  not  so  striking,  as  in  the  case  of  the  toxin-antitoxin  mixtures. 
This,  however,  cannot  be  surprising,  if  it  is  borne  in  mind  that  con- 
ditions here  are  much  more  complex.  We  have  pointed  out  before 
that  an  apparent  paradox  results  when  a  constant  quantity  of 
bacteria  is  treated  with  increasing  quantities  of  agglutinin  (page  111), 
but  we  have  shown  that  this  finds  a  ready  explanation  on  the  basis 
of  the  existence  of  so-called  partial  agglutinins  which  are  "tuned" 
to  different  agglutinable  molecules  in  the  bacteria,  and  that  as  a 
consequence  the  apparent  binding  power  of  bacteria  for  their  agglu- 
tinins may  be  perfectly  colossal.  Under  such  circumstances  one 


124  THE  SIDE-CHAIN  THEORY 

could  hardly  expect  to  throw  out  of  action  all  the  agglutinable  groups 
in  a  given  quantity  of  bacteria  by  treating  these  with  a  corresponding 
agglutinin,  even  in  very  large  amount.  But  notwithstanding  this 
difficulty  Neisser  and  Lubowski  obtained  some  sera  by  immunizing 
with  such  mixtures,  which  contained  no  agglutinins  at  all.  This,  to 
be  sure,  was  exceptional;  but  they  could  show,  nevertheless,  that  in 
a  series  of  experiments  the  subsequent  agglutinative  value  averaged 
only  1  to  106  as  compared  with  1  to  1093  in  the  control  animals, 
viz.,  in  those  which  had  been  injected  with  non-agglutinated 
organisms. 

On  the  basis  of  Ehrlich's  theory  the  appearance  of  the  so-called 
natural  antibodies  in  the  serum  can  now  also  be  accounted  for  in 
a  ready  manner.  Since  the  antibodies  are  not  formed  de  now,  but 
merely  represent  normal  molecular  complexes  of  the  body  cells,  it 
can  hardly  be  surprising  that  once  in  a  while,  even  in  the  course  of 
normal  events,  some  of  these  side  chains  will  be  cast  off,  although  no 
bacteria  or  their  toxins  may  have  entered  the  body.  That  the  anti- 
bodies, moreover,  which  result  on  immunization  with  foreign  cells 
or  cell  products  should  be  specific,  is  a  necessary  consequence,  if  we 
accept  the  view  that  antibody  production  presupposes  the  existence 
of  a  special  affinity  between  the  haptophoric  groups  of  antigen  and 
antibody.  The  remarkable  point  in  this  connection  indeed  is  not  so 
much  the  fact  that  the  injection  of  a  toxin  should  give  rise  to  an 
antitoxin,  or  of  bacteria  to  corresponding  lysins  or  cytotoxins,  but 
that  so  many  varieties  of  antibodies  should  be  possible  for  a  given 
animal. 

On  the  basis  of  Ehrlich's  theory  we  are  forced  to  conclude  that 
the  cells  of  the  body  collectively  must  contain  at  least  as  many 
different  types  of  side  chains  preformed  as  the  number  of  different 
antibodies  that  can  be  theoretically  obtained  from  a  given  animal, 
and  vice  versa.  This,  however,  does  not  seem  altogether  likely,  if 
we  bear  in  mind  the  innumerable  varieties  of  antibodies  that  can 
actually  be  produced.  I  would  only  recall  the  possibility  of  obtain- 
ing specific  precipitins  to  the  albumins  of  almost  all  the  different 
types  of  animals,  then  again  the  production  of  agglutin  ns  not  only 
to  different  species  of  bacteria,  but  even  to  different  strains  of  a 
single  species,  etc.  But  it  seems  to  me  that  even  though  we  accept 
Ehrlich's  theory  in  its  essential  points  that  we  need  not  suppose 
the  existence  of  such  an  enormous  variety  of  receptors  as  occurring 


FORMATION  OF  ANTIBODIES  125 

preformed.  It  would  seem  perfectly  plausible  that  though  some 
of  the  receptors,  which  we  meet  with  as  antibodies,  may  actually 
exist  preformed,  that  others  are  developed  only  when  certain  anti- 
gens are  brought  in  contact  with  certain  cells,  and  in  consequence  of 
a  special  "Bildungsreiz."  Experiments  in  this  direction  have,  so 
far  as  my  knowledge  goes,  not  yet  been  made,  but  it  should  be 
possible  to  test  the  hypothesis  just  set  forth.  If  my  surmise  were 
correct  a  still  more  plausible  explanation  of  the  specificity  of  the 
antibodies  would  thus  be  afforded. 

Before  concluding  the  present  chapter  one  more  point  may  yet 
be  appropriately  considered,  viz.,  the  question  why  those  poisons 
which  we  can  prepare  in  pure  form  in  the  chemical  laboratory,  and 
whose  structural  composition  is  known,  such  as  the  various  alka- 
loids, glucosides,  alcohols,  etc.,  do  not  give  rise  to  antibody  formation. 
The  fundamental  reason  for  this  differing  behavior  according  to 
Ehrlich  lies  in  the  fact  that  the  true  antigens  are  chemically  bound, 
and  that  chemical  interaction  between  antigen  and  cell  receptor 
takes  place  because  the  bodies  in  question  are  structurally  closely 
allied  to  the  true  foodstuffs.  The  majority  of  poisons  of  the  chemi- 
cal laboratory,  on  the  other  hand,  are  not  taken  up  by  the  cells  in 
virtue  of  the  existence  of  a  special  chemical  affinity,  but  merely  in 
consequence  of  physical  influences. 

This  is  well  shown  in  the  following  experiment.  After  it  had  been 
discovered  by  Ehrlich  and  Overton  that  the  injection  of  various 
anilin  dyes  leads  to  their  storage  in  certain  tissues  of  the  body,  and 
that  this  storage  is  due  to  the  presence  in  these  tissues  of  certain 
lipoids  which  act  as  solvents  for  the  pigments  in  question,  Hans 
Meyer  and  Overton  could  demonstrate  that  the  strength  of  various 
narcotics  is  not  dependent  upon  their  chemical  composition,  but 
upon  their  coefficient  of  distribution  which  regulates  their  distri- 
bution between  the  blood  plasma  and  the  lipoids  of  the  brain.  This 
is  well  shown  in  the  table  on  page  126,  which  is  taken  from  Baum. 
The  first  column  of  figures  represents  the  coefficient  of  distribution 
of  the  various  narcotics,  as  calculated  for  water  on  the  one  hand, 
and  fat  on  the  other  (calculated  for  olive  oil),  while  the  figures  of 
the  second  column  indicate  the  amount  of  the  substances  per  liter, 
expressed  in  fractions  of  the  corresponding  normal  solutions,  which 
are  just  sufficient  to  produce  narcosis  in  the  test  animal  (usually 
frog  larvae);  this  is  termed  the  threshold  of  action.  By  comparing 


126  THE  SIDE-CHAIN  THEORY 

the  two  columns  it  will  be  noted  that  notwithstanding  the  wide 
variations  in  the  chemical  structure  of  the  different  narcotics,  their 
effect  is  evidently  dependent  upon  purely  physical  conditions,  viz., 
the  coefficient  of  distribution. 

Coefficient  of  distribution       Threshold  of  action  ex- 
Concentration  in  fat  pressed  in  fractions  of 
Narcotic.                                     =  Concentration  in  water        the  normal  solutions. 

Trional 4.46  0.0018 

Tetronal 4.04  0.0013 

Butylchoralhydrate      ...  1.59  0.002 

Sulfonal 1.11  0.006 

Bromalhydrate 0 . 66  0 . 002 

Triazetin 1.30  0.01 

Diacetin 0.23  0.015 

Chloralhydrate 0.22  0.02 

Ethylurethane 0.14  0.04 

Monacetin 0.06  0.05 

Methylurethane      ....  0.04  0.4 

In  accord  with  this  view  regarding  the  action  of  the  majority  of 
the  chemical  poisons  upon  the  cells  of  the  body,  is  also  the  fact, 
that  these  substances  can  again  be  extracted  from  the  cells  by  the 
use  of  appropriate  solvents,  which,  of  course,  would  not  be  possible 
if  chemical  union  had  taken  place. 

We  may  thus  sum  up  by  saying  that  only  those  substances  can 
possess  antigenic  properties  which  are  capable  of  entering  into 
chemical  union  with  the  cells,  but  that  in  addition  a  special  "Bin- 
dungsreiz"  must  be  exercised  upon  the  cell,  which  is  peculiar  to 
the  antigens. 


CHAPTER  IX 
THE  DIFFERENT  TYPES  OF  IMMUNITY 

WE  have  seen  in  the  foregoing  chapter  how  satisfactorily  Ehrlich's 
theory  accounts  for  the  formation  and  specific  action  of  the  anti- 
bodies, and  thus  for  the  origin  and  mode  of  action  of  some  of  the 
most  important  defensive  factors  of  the  animal  body.  Upon  this 
basis  we  may  now  also  take  up  for  consideration  some  of  the  more 
general  aspects  of  the  problem  of  immunity. 

When  we  speak  of  immunity  in  the  biological  sense,  we  under- 
stand thereby  the  existence  of  a  certain  resistance  toward  dele- 
terious influences.  This  may  be  directed  against  a  large  number 
of  factors,  such  as  the  action  of  various  drugs  and  chemicals,  the 
harmful  effect  of  atmospheric  conditions,  attack  by  other  animals, 
various  degenerative  influences  arising  from  within  the  body,  infec- 
tions with  vegetable  or  animal  parasites  and  the  absorption  of  their 
products  of  metabolism  or  degeneration,  etc.  From  a  medical 
standpoint,  of  course,  these  latter  influences  interest  us  particularly, 
and  in  the  following  pages  we  shall  devote  our  attention  to  the 
subject  of  immunity  from  this  standpoint  more  especially. 

Natural  Immunity. — The  very  fact  that  animal  life  is  possible  at 
all,  surrounded  as  we  are  by  organisms  which  under  certain  con- 
ditions can  invade  the  body  and  cause  its  destruction,  shows  in 
itself  that  every  individual  must  possess  a  certain  degree  of  natural 
immunity.  Staphylococci  are  thus  found  not  only  on  the  outer 
surface,  but  even  in  the  deeper  layers  of  the  skin  without  giving  rise 
to  any  disturbance;  pathogenic  pneumococci,  streptococci,  and  even 
diphtheria  bacilli  may  be  present  in  the  fauces  without  producing  dis- 
ease; the  intestinal  canal  is  inhabited  by  untold  millions  of  bacteria, 
some  of  them  of  pathogenic  character,  which  apparently  produce 
no  deleterious  effects,  and  so  on.  Apparently  the  individual  who 
normally  harbors  all  these  various  organisms  is  immune  to  the 
corresponding  infections. 

The  picture,  however,  changes  very  materially,  if  in  some  manner 


128  THE  DIFFERENT  TYPES  OF  IMMUNITY 

a  break  in  the  continuity  of  the  epithelial  lining  of  the  outer  or  inner 
surfaces  of  the  body  occurs.  A  surface  bruise  may  be  followed  by 
the  formation  of  an  abscess,  an  injury  to  the  nose  may  lead  to 
meningitis,  the  irritation  of  the  gall-bladder  by  a  calculus  may  be 
followed  by  cholecystitis.  The  gardener  or  the  stable  man  may 
have  his  hands  soiled  by  material  containing  tetanus  bacilli  with- 
out any  harm,  while  the  infliction  of  a  trifling  wound  may  lead  to 
fatal  lockjaw.  Evidently  the  immunity  to  certain  diseases  which 
one  would  infer  from  the  presence  of  the  corresponding  pathogenic 
organisms  on  the  surface  of  the  body  in  the  absence  of  symptoms 
of  disease  is  only  apparent. 

All  that  we  can  infer  from  such  observations  is  that  the  surface 
epithelium  shows  a  certain  degree  of  resistance  to  infection,  i.  e., 
that  in  a  certain  sense  at  least  it  is  immune.  Numerous  observa- 
tions go  to  show,  as  a  matter  of  fact,  that  local  conditions  play  an 
important  role  in  determining  the  degree  of  resistance  to  infection. 
It  has  thus  been  demonstrated  that  certain  organisms  can  only 
infect  when  they  are  introduced  in  a  certain  manner,  while  others 
can  do  so  from  practically  any  point.  The  pathogenic  cocci  and 
plague  bacillus  are  examples  of  the  latter  kind,  while  the  dysentery 
bacillus,  the  cholera  vibrio,  and  certain  meat-poisoning  bacilli  require 
a  special  portal  of  entry.  We  may  then  conclude  that  the  body 
possesses  virtually  no  tissue  immunity  toward  the  first  and  a  fairly 
high  grade  of  immunity  toward  the  second  order.  Quite  in  accord- 
ance with  these  observations  is  the  fact  that  in  certain  animals 
tetanus  can  be  produced  only  by  intracerebral  injection  of  the 
corresponding  toxin,  while  in  others  the  disease  develops,  no  matter 
what  the  character  of  the  tissue  may  be  in  which  the  injection  is 
made. 

The  same  point  is  also  well  illustrated  by  the  remarkable  predi- 
lection which  certain  organisms  have  for  certain  organs,  when  once 
they  have  passed  the  outer  epithelial  barriers.  If  young  rabbits 
are  thus  injected  intravenously  with  cholera  vibrios  they  die  after  a 
few  days,  and  post  mortem  the  organisms  are  found  in  large  num- 
bers in  the  intestinal  mucosa,  while  the  blood  and  remaining  organs 
are  sterile  (providing,  of  course,  that  the  number  injected  has  not 
been  unduly  large).  Evidently  the  intestinal  mucous  membrane 
offers  little  or  no  resistance  to  the  cholera  vibrio,  while  the  other 
tissues  show  a  considerable  degree  of  immunity.  Well  known,  also, 


CLASS  IMMUNITY  129 

is  the  marked  affinity  which  exists  between  the  meningococcus  and 
the  meninges,  of  the  pneumococcus  for  pulmonary  tissue,  of  strepto- 
cocci for  serous  membranes,  of  the  typhoid  bacillus  for  lymphoid 
structures,  etc.;  while  the  other  tissues  show  a  more  or  less  well- 
defined  immunity.  Evidently  the  degree  of  resistance  or  immunity 
which  the  animal  offers  to  infection  depends  both  upon  the  nature 
of  the  organism  and  the  route  by  which  it  is  introduced. 

If  infection,  by  what  we  may  term  a  natural  route,  is  excluded, 
then  there  will  be  an  apparent  immunity,  at  least,  to  the  organism 
in  question.  For  practical  purposes  this  type  of  immunity  may, 
indeed,  be  regarded  as  absolute.  But  that  it  is  not  so  of  necessity 
can  in  some  instances  be  demonstrated  by  introducing  the  organism 
through  channels  by  which  natural  infection  would  not  be  likely 
to  occur.  In  the  human  being,  typhoid  infection  will  thus  almost 
always  develop  by  way  of  the  intestinal  canal.  In  most  of  our  labor- 
atory animals,  infection  by  this  channel  is  impossible,  and  we  might 
accordingly  regard  them  as  immune.  That  this  is  only  apparently 
the  case,  however,  can  be  readily  shown  by  injecting  the  organisms 
intraperitoneally,  when  a  fatal  infection  can  be  produced  at  will. 
The  fact  remains,  nevertheless,  that  the  various  animals  in  their 
natural  state  do  not  contract  typhoid  fever,  although  they  must 
be  exposed  to  infection  on  many  occasions.  They  may  hence  be 
regarded  as  practically  immune.  Many  instances  of  immunity  no 
doubt  are  dependent  upon  such  causes,  viz.,  upon  the  existence 
of  immunity  of  those  tissues  by  which  natural  infection  would 
ordinarily  occur. 

Class  Immunity. — Generally  speaking,  the  natural  susceptibility 
to  infection  by  microorganisms  differs  with  the  different  classes 
of  animals,  with  different  genera,  with  different  species,  and  even 
with  different  varieties  and  individuals.  We  accordingly  recognize 
a  natural  class  immunity,  a  natural  generic  and  species  immunity, 
natural  race  immunity,  and  individual  immunity.  Class  immunity 
is  especially  interesting  because  it  presents  examples  of  absolute 
immunity,  under  natural  conditions  at  least,  which  is,  after  all, 
exceedingly  rare.  The  immunity  of  cold-blooded  animals  toward 
the  majority  of  those  organisms  which  are  pathogenic  for  warm- 
blooded animals  belongs  to  this  order.  But  even  here  the  immunity 
is  sometimes  only  relative  and  apparent.  The  frog  is  thus  naturally 
insusceptible  to  anthrax,  and  the  injection  of  large  numbers  of  such 
9 


130  THE  DIFFERENT  TYPES  OF  IMMUNITY 

organisms  will  under  ordinary  conditions  produce  no  deleterious 
results.  If,  however,  the  animals  are  kept  at  a  temperature  at  which 
anthrax  bacilli  can  readily  grow,  infection  promptly  takes  place. 
Conversely,  Pasteur  found  that  it  is  possible  to  infect  chickens 
with  anthrax,  by  refrigeration,  whereas  normally  the  animal  is 
immune. 

Toward  the  leprosy  bacillus  there  is  apparently  an  absolute 
immunity  not  only  on  the  part  of  the  cold-blooded  animals,  but 
also  of  the  vertebrates,  with  the  exception  of  man  and  possibly 
of  certain  monkeys. 

Generic  Immunity. — As  examples  of  generic  immunity  we  may 
mention  the  resistance  of  man  to  the  common  organisms  which  are 
pathogenic  for  the  lower  vertebrates,  and  vice  versa. 

Species  Immunity. — Species  immunity  is  illustrated  by  the  resist- 
ance of  dogs,  pigs,  and  rats  to  the  anthrax  bacillus,  while  cattle, 
sheep,  and  most  of  the  common  laboratory  animals  are  quite  sus- 
ceptible to  infection  with  this  organism.  Cattle  plague  (rinderpest), 
swine  plague  (Schweinerotlauf),  sympathetic  anthrax  (Rauschbrand), 
chicken  cholera,  etc.,  do  not  affect  man  under  normal  conditions, 
while  animals  are  naturally  immune  to  infection  with  the  cholera 
vibrio,  the  meningococcus,  the  typhoid  bacillus,  the  gonococcus 
the  treponema  pallidum,  as  well  as  to  such  diseases  as  scarlatina, 
measles,  yellow  fever,  poliomyelitis,  etc. 

Racial  Immunity. — Racial  immunity  is  exemplified  by  the  relatively 
high  degree  of  resistance  of  Algerian  sheep  to  anthrax,  to  which 
our  own  domestic  sheep  are  very  prone.  Black  rats  are  more  resist- 
ant to  anthrax  than  gray  rats  and  gray  rats  more  so  than  white 
rats.  The  same  point  is  also  well  shown  in  the  remarkable  difference 
in  the  susceptibility  of  different  races  to  such  diseases  as  measles, 
smallpox,  tuberculosis,  etc. 

Individual  Variations  in  Susceptibility. — The  occurrence  of  indi- 
vidual variations  in  the  susceptibility  to  various  diseases,  further, 
is  so  well  known  as  hardly  to  require  special  mention.  During 
epidemics  of  cholera,  smallpox,  diphtheria,  yellow  fever,  typhoid 
fever,  influenza,  etc.,  this  is  particularly  noticeable.  There  are  then 
always  some  persons  who  escape  infection  even  though  they  have 
been  freely  exposed,  and  among  those  which  develop  the  diseases 
in  question  there  are  some  in  whom  the  malady  runs  a  mild  course, 
while  others  are  fatally  stricken;  in  some  we  see  a  remarkable 


ACQUIRED  IMMUNITY  131 

tendency  to  complications,  while  others  recover  without  any  unto- 
ward incident,  and  so  on.  We  must  accordingly  conclude  that  some 
individuals  are  naturally  immune  to  certain  infections  and  that 
even  among  those  who  are  attacked  there  must  be  marked  quan- 
titative variations  in  resistance. 

Acquired  Immunity. — The  different  types  of  immunity  which  have 
been  briefly  considered  above  have  one  point  at  least  in  common — 
namely,  the  fact  that  they  exist  under  natural  conditions,  and  we 
hence  speak  of  immunity  of  this  order  as  natural  immunity.  With- 
out entering  into  a  discussion  of  the  possible  etiological  factors 
which  may  have  been  operative  in  the  production  of  this  type  of 
immunity,  we  may  emphasize  that  it  apparently  does  not  depend 
upon  a  process  of  active  immunization,  viz.,  upon  the  introduction 
either  of  the  pathogenic  organism  or  its  product.  This  is  in  marked 
contradistinction  to  another  type  of  immunity  which  is  directly 
dependent  upon  these  very  factors  and  which  we  accordingly  speak 
of  as  acquired  immunity. 

It  has  long  been  recognized  that  individuals  who  have  once  passed 
through  certain  diseases,  such  as  smallpox,  chicken-pox,  scarlatina, 
measles,  mumps,  whooping  cough,  typhoid  fever,  typhus  fever,  yellow 
fever,  and  Asiatic  cholera,  are  subsequently  immune  either  abso- 
lutely, or  to  a  very  considerable  extent.  The  recognition  of  this  fact 
has  been  of  the  greatest  importance,  for  it  forms  the  basis  of  our 
modern  attempts  to  create  an  artificial  immunity  to  different  dis- 
eases, or  if  not  an  actual  immunity,  then  at  least  increased  resistance 
by  the  purposeful  introduction  of  the  corresponding  infecting  agent 
in  such  form  as  not  to  expose  the  individual  to  the  dangers  of  natural 
infection.  We  may  thus  distinguish  between  an  artificially  acquired 
immunity  and  what  we  may  appropriately  term  accidentally  acquired 
immunity. 

The  discovery  of  the  possibility  of  producing  immunity  artifi- 
cially we  owe  to  Jenner,  who  first  showed  that  by  "vaccinating" 
individuals  with  smallpox  virus  which  had  been  attenuated  by 
passage  through  cattle,  protection  against  the  dreaded  malady 
could  be  secured  (1798).  Although  the  causative  agent  of  smallpox 
was  unknown,  Pasteur  subsequently  recognized  that  the  principle  of 
vaccination  lies  in  the  production  of  the  disease  in  an  attenuated  form. 
The  thought  hence  suggested  itself  to  him  that  the  same  principle 
might  be  adapted  to  the  prevention  of  bacterial  diseases  also,  and  by 


132  THE  DIFFERENT  TYPES  OF  IMMUNITY 

experimentation  in  this  direction  he  laid  the  foundation  of  our 
modern  vaccine  therapy,  which  finds  its  most  important  expression, 
so  far  as  human  pathology  is  concerned,  in  the  curative  (sc.,  preven- 
tive) treatment  of  rabies,  and  in  the  prophylactic  vaccination  against 
typhoid  fever.  In  the  laboratory  it  has  further  led  to  the  recognition 
of  the  fact  that  even  though  immunity  cannot  be  produced  against 
all  pathogenic  organisms  by  vaccination,  it  is  at  least  possible  to 
bring  about  a  marked  increase  in  resistance,  and  by  applying  this 
principle  to  other  infectious  disease?,  to  which  man  is  subject,  a 
radical  advance  in  the  rational  treatment  of  these  maladies  has 
been  achieved  (see  section  on  Vaccine  Therapy). 

Antitoxic  Immunity. — In  a  previous  chapter  we  have  seen  that  some 
pathogenic  organisms  injure  the  host  into  which  they  have  been 
introduced  through  the  products  of  their  metabolism  or  degenera- 
tion, insofar  as  these  are  of  toxic  character,  while  their  infectious- 
ness  may  be  of  a  very  low  order.  Others  produce  a  harmful  effect 
directly  in  consequence  of  their  high  grade  of  infectiousness,  even 
though  they  do  not  give  rise  to  toxic  products,  while  in  still  other 
cases  we  see  both  factors  variously  combined.  Evidently,  then, 
the  existence  of  a  natural  immunity,  or  of  immunity  brought  about 
as  a  consequence  of  infection,  may  manifest  itself  either  as  a  resist- 
ance of  variable  degree  against  the  development  of  microorganisms 
in  the  body  of  the  infected  animal,  or  as  a  resistance  against  bacterial 
toxins,  endotoxins,  aggressins,  etc.,  or  it  may  be  directed  against 
both.  It  is,  hence,  appropriate  to  speak  of  antitoxic  immunity  on 
the  one  hand,  and  antibacterial  immunity  on  the  other. 

As  an  example  of  natural  antitoxic  immunity  we  may  mention 
the  natural  resistance  which  the  alligator  offers  to  the  action  of 
tetanus  toxin,  while  the  steadily  increasing  resistance  to  diphtheria 
toxin  manifested  by  a  horse  undergoing  corresponding  immuniza- 
tion may  serve  as  an  illustration  of  acquired  antitoxic  immunity. 
The  natural  resistance  of  rats  and  dogs  to  anthrax,  on  the  other  hand, 
is  of  antibacterial  character,  as  is  also  the  immunity  or  increased 
resistance,  at  any  rate,  which  results  on  vaccination  with  the  same 
organism  in  otherwise  susceptible  animals,  such  as  sheep,  guinea- 
pigs,  and  mice. 

If  an  individual  becomes  immune  to  a  given  organism  or  its  toxic 
products  as  a  result  of  infection  or  vaccination,  in  consequence  of 
his  own  efforts,  as  it  were,  we  speak  of  active  immunity,  while  immu- 


MECHANISM  OF  DIFFERENT  TYPES  OF  IMMUNITY     133 

nity  which  results  from  the  transference  of  protecting  substances 
from  an  immune  animal  to  a  non-immune  individual  is  designated 
as  passive  immunity.  The  difference  between  the  two  is  well  illus- 
trated, if  we  compare  the  recovery  of  a  diphtheria  patient  without 
treatment,  with  the  recovery  of  one  which  follows  as  a  consequence 
of  the  administration  of  antitoxin. 

In  the  first  instances  the  patient  recovers  because  he  succeeds  in 
forming  enough  antitoxin  in  his  own  body  to  neutralize  the  toxin 
produced  by  the  invading  organism,  pending  the  destruction  of  the 
bacteria  by  other  means,  while  in  the  second  the  patient  is  protected 
against  the  deleterious  effects  of  the  toxin  through  the  introduction 
of  the  corresponding  antitoxin  from  without.  The  possibility  of 
passive  immunization  is,  of  course,  of  the  utmost  importance  as 
successful  serum  therapy  during  the  actual  progress  of  a  disease  is 
dependent  upon  this  principle,  while  active  immunization  in  the 
nature  of  things  forms  the  basis  of  prophylactic  vaccination. 

Mechanism  of  Different  Types  of  Immunity. — If  now  we  turn  to  a 
consideration  of  the  mechanism  which  underlies  the  different  types 
of  immunity,  as  just  outlined,  various  possibilities  suggest  themselves. 

Aside  from  those  factors  which  render  the  actual  invasion  difficult 
if  not  impossible,  such  as  the  character  of  the  epithelial  covering 
and  the  nature  of  the  secretions  which  are  poured  out  upon  the 
epithelial  surfaces,  the  chemical  and  physical  characteristics  of  the 
medium  in  which  the  organism  finds  itself  after  invasion  has  taken 
place  are  of  necessity  determinative  for  the  question  whether  infec- 
tion will  or  will  not  occur.  These  factors,  of  course,  may  be  entirely 
independent  of  any  direct  bactericidal  action  of  the  body  cells  and 
juices  per  se,  and  have  to  do  simply  with  the  character  of  the  environ- 
ment, viewed  as  a  culture  medium  for  the  organism  in  question. 

We  know  that  certain  organisms  can  develop  successfully  outside 
of  the  body  only,  if  the  temperature,  the  reaction  of  the  culture 
medium,  and  its  chemical  composition  are  of  a  definite  character. 
The  remarkable  fastidiousness  in  this  respect  of  such  organisms  as 
the  gonococcus  and  the  influenza  bacillus  is  well  known.  It  is  accord- 
ingly quite  conceivable  that  infection  with  certain  organisms  cannot 
occur  because  of  the  unfavorable  character  of  some  factors  of  this 
order  within  the  body.  The  effect  of  temperature  in  this  respect 
is  thus  well  shown  in  the  case  of  cold-blooded  animals,  like  the  frog, 
which  is  naturally  immune  to  infection  with  the  anthrax  bacillus, 


134  THE  DIFFERENT  TYPES  OF  IMMUNITY 

but  which  loses  its  resistance  when  kept  at  a  temperature  at  which 
the  organism  will  normally  develop  outside  of  the  body.  Conversely 
it  has  been  noted  that  frogs  which  under  natural  conditions,  i.  e., 
at  low  temperature,  readily  fall  a  prey  to  infection  with  the  bacillus 
ranicida,  are  immune  to  the  same  organism  if  kept  at  a  temperature 
of  25°  C.  Of  the  same  order,  no  doubt,  is  the  immunity  of  chickens 
to  anthrax,  which  disappears  when  the  animals  are  kept  immersed 
in  water  of  25°  C.,  their  normally  high  temperature  being  reduced 
in  this  manner.  In  cases  such  as  these  the  modus  operandi  of  the 
temperature  changes  upon  immunity  or  infection  seems  relatively 
simple,  while  in  others  it  is  certainly  of  a  more  complex  order. 

It  is  thus  a  well-known  fact  that  man  and  other  animals  after 
exposure  to  cold  are  more  prone  to  infection  with  a  number  of  different 
organisms,  which  find  their  optimum  condition  for  growth  at  the 
normal  temperature  of  the  body.  The  underlying  causes  of  the 
change  in  resistance  in  such  cases  are  apparently  different,  but  what 
the  mechanism  is  we  do  not  know.  We  can  readily  imagine,  however, 
that  functional  disturbances  may  be  set  up  in  the  macro  organism 
by  the  cold  which  in  some  manner  operates  to  the  advantage  of  the 
microorganism.  It  is  not  excluded,  of  course,  that  in  the  instances 
of  immunity  mentioned  above,  something  similar  may  not  also  be 
operative,  but  the  simple  explanation  that  has  been  offered  cannot 
be  overlooked. 

Athreptic  Immunity. — In  other  cases  the  resistance  to  infection 
may  be  referable  to  the  existence  of  unfavorable  conditions  of 
nutrition.  A  number  of  observations  have  taught  us  that  certain 
organisms  require  certain  specific  foodstuffs  for  their  development, 
in  addition  to  others  which  are  necessary  to  all  forms  of  life  of  that 
order,  and  unless  these  are  present,  successful  growth  cannot  take 
place  and  immunity  would  accordingly  result.  Immunity  of  this 
type  is  spoken  of  as  athreptic  immunity. 

Ehrlich  first  suggested  this  term  to  denote  the  peculiar  behavior 
of  mouse  cancer  when  transplanted  into  rats.  At  first  active  growth 
takes  place,  so  that  at  the  end  of  eight  to  ten  days  the  size  of  the 
tumor  does  not  differ  from  control  tumors  in  mice.  After  that, 
however,  further  growth  ceases  and  resorption  takes  place.  If, 
now,  i.  e.,  at  a  time  when  active  growth  no  longer  occurs  in  rat  A  a 
transplant  be  made  to  another  rat  B  the  graft  does  not  develop.  But 
if  a  mouse  be  inoculated  instead,  active  growth  takes  place,  and 


ANTIAGGRESSlN  IMMUNITY  135 

if  from  this  a  transplant  is  made  to  rat  B  a  tumor  develops  as  in 
rat  A.  To  explain  this  peculiar  behavior  Ehrlich  suggested  that 
some  specific  substance  which  we  may  call  X,  and  which  is  supposedly 
found  only  in  the  body  of  the  mouse,  and  which  is  essential  to 
the  growth  of  the  mouse  cancer,  is  transferred  to  rat  A  when  the 
first  transplantation  is  made.  As  long  as  a  supply  of  this  substance 
is  available  the  cancer  cell  can  multiply  and  make  use  of  the  usual 
foodstuffs  of  the  organism  of  the  rat.  As  soon  as  this  is  exhausted, 
however,  further  development  is  not  possible,  and  if  at  this  time 
rat  B  is  inoculated  no  growth  occurs  because  the  specific  growth 
stuff  X  is  absent.  If  a  transplant  be  made  back  to  a  mouse,  how- 
ever, X  is  again  supplied  and  a  transfer  to  rat  B  will  then  again 
lead  to  successful  growth  until  X  is  again  used  up.  The  immunity 
of  the  rat  to  the  mouse  cancer  is  thus  evidently  dependent  upon  an 
athrepsia,  i.  e.,  an  absence  of  a  specific  substance  which  is  essential 
to  the  growth  of  the  mouse  tumor  cells.  This  concept  of  a  certain 
form  of  tumor  immunity  is  theoretically,  at  least,  applicable  to  certain 
types  of  antibacterial  immunity  also,  even  though  the  experimental 
basis  for  such  an  assumption  has  not  yet  been  supplied. 

We  know,  of  course,  that  certain  organisms  can  be  grown  outside 
of  the  body  only,  if  certain  special  substances  are  supplied  and  that 
in  their  absence  growth  ceases.  A  familiar  example  is  furnished 
by  the  influenza  bacillus.  If  this  is  transplanted  from  hemorrhagic 
sputum  to  ordinary  culture  media  a  certain  amount  of  growth  is 
at  first  obtained,  but  unless  hemoglobin  is  artificially  supplied  to 
the  subcultures  the  organism  soon  dies  out.  We  may  accordingly 
imagine  that  certain  animals  are  immune  to  infection  with  certain 
organisms  because  the  macroorganism  does  not  supply  all  those 
substances  which  are  essential  to  the  growth  of  the  microorganism, 
but,  as  just  stated,  we  do  not  as  yet  know  what  those  substances 
are,  and  we  do  not  know  against  what  organisms  an  athreptic 
immunity  exists.  We  merely  recognize  the  possibility  and  must 
reckon  with  it  in  our  discussion  of  the  subject. 

Antiaggressin  Immunity. — Another  factor  which  must  be  considered 
in  connection  with  the  question  regarding  the  mechanism,  which 
is  operative  in  the  production  of  antibacterial  immunity,  is  the 
possibility  that  the  organism,  which  has  found  its  way  into  the 
body,  may  be  devoid  of  all  aggressivity,  and  that  it  hence  falls  an 
easy  prey  to  the  normal  defensive  mechanism  of  the  macroorganism. 


136  THE  DIFFERENT  TYPES  OF  IMMUNITY 

Here  also  our  knowledge  is  as  yet  very  meager,  but  it  would  seem 
that  this  possibility  actually  exists.  In  the  case  of  the  anthrax 
bacillus,  for  example,  it  has  been  ascertained  that  by  suitable  methods 
the  organism  can  be  deprived  of  its  power  to  form  capsules  and 
that  such  strains  are  then  no  longer  capable  of  producing  infection. 
We  have  seen  before  that  this  organism  owes  its  infectiousness 
to  a  large  extent  to  its  ability  to  surround  itself  with  a  capsule, 
and  that  when  once  encapsulated  it  is  no  longer  open  to  successful 
attack  by  the  phagocytes.  It  is  thus  easy  to  see  why  an  animal 
should  prove  immune  to  infection  when  an  organism  is  introduced 
which  depends  for  its  existence  in  its  new  environment  upon  aggres- 
sive factors  of  this  order  and  is  incapable  of  developing  them. 

While  immunity  of  this  type  would  depend  upon  lack  of  aggres- 
sivity  in  the  more  general  sense  on  the  part  of  an  organism,  there  is 
evidence  to  show  that  immunity  may  also  be  due  to  the  same  factor 
in  the  more  restricted  sense  of  Bail,  viz.,  upon  an  inability  of  the 
organism  to  overcome  the  normal  defensive  factors  by  the  secretion 
or  liberation  of  soluble  aggressins.  This  is  well  illustrated  in  the 
case  of  the  pigeon,  which  is  markedly  immune  to  anthrax  even 
though  its  serum  per  se  is  not  bactericidal  for  this  organism.  Upon 
the  addition  of  leukocytes,  however,  it  becomes  so,  and  against 
this  combination  an  amount  of  anthrax  aggressin  is  powerless  which 
would  suffice  to  overcome  the  bactericidal  power  of  a  corresponding 
serum-leukocyte  mixture  taken  from  a  guinea-pig  which  itself  is 
markedly  antiaggressive  in  its  action.  Of  the  manner  in  which 
this  effect  is  produced,  however,  we  know  nothing. 

We  denote  this  type  of  immunity  as  antiaggressin  immunity  merely 
to  express  the  fact  that  it  depends  upon  factors  which  are  not  of 
a  bactericidal  nature,  but  which  prevent  the  development  of  those 
aggressive  functions  upon  which  certain  organisms  depend  for  their 
existence,  after  invasion  of  the  body  has  taken  place.  The  animal 
is  immune  not  because  it  has  stronger  bactericidal  forces  either  in 
its  serum  or  its  cells,  not  because  it  can  prevent  the  animalization 
of  the  invading  organisms,  not  because  of  any  antitoxic  mechanism, 
but  because  the  organisms  for  some  reason  find  themselves  incapable 
of  exercising  their  special  aggressive  forces.  But  of  the  reasons  why 
this  should  be  so  in  one  animal  and  not  in  another,  we  know  nothing. 

While  the  existence  of  an  antiaggressin  immunity  in  the  special 
sense  of  Bail,  as  just  outlined,  has  thus  far  been  established  only 


ANTIBACTERIAL  IMMUNITY  137 

in  a  single  one  of  the  naturally  immune  animals,  it  is  not  unlikely 
that  the  same  mechanism  may  be  operative  in  the  production  of 
natural  immunity  in  others,  and  especially  in  connection  with  those 
organisms  which,  like  the  anthrax  bacillus,  are  characterized  by  a 
high  degree  of  infectiousness  and  a  low  grade  of  toxicity.  In  the 
case  of  some  of  these,  it  probably  also  plays  a  role  in  the  develop- 
ment of  an  acquired  immunity. 

Antibacterial  Immunity. — In,  the  majority  of  infections  with  the 
semiparasites,  on  the  other  hand,  the  acquired  immunity  is  not 
antiaggressive,  but  bactericidal  in  character,  and  since  bactericidal 
influences  may  be  exercised  either  by  the  serum  alone  or  the  leuko- 
cytes alone,  or  by  both  in  combination,  the  resultant  immunity 
may,  theoretically  at  least,  be  due  to  an  exaggerated  functional 
activity  of  either  one  or  both  of  these  factors.  It  is  not  my  purpose 
at  this  place  to  enter  into  a  discussion  of  the  question  which  one  of 
the  two  is  really  the  primum  movens  in  the  production  or  existence 
of  antibacterial  immunity.  I  would  merely  recall  that  for  many 
years,  immunity  students  were  divided  into  two  opposing  factions, 
viz.,  the  humoral  school,  led  by  Pfeiffer,  and  the  older  phagocytic 
school,  represented  by  MetschnikofF,  whose  respective  standpoints 
seemed  for  a  long  time  irreconcilable  the  one  with  the  other.  At 
the  present  time  the  original  sharp  lines  between  the  two  schools 
have  fallen,  and  we  recognize  that  there  is  an  intimate  interrelation- 
ship between  the  cellular  and  the  humoral  defences,  that  the  two 
supplement  one  another  and  that  neither  alone  should  be  viewed 
as  sufficient  to  protect  an  animal  against  infection  and  its  con- 
sequences. But  while  recognizing  the  importance  of  both,  we  must 
also  admit  that  neither  the  one  nor  the  other  seems  to  be  solely 
responsible  for  the  development  of  an  acquired  immunity. 

There  can  be  no  doubt  that  as  a  result  of  infection  or  vaccination, 
corresponding  bacteriolytic  amboceptors  are  formed  in  large  quantity 
and  that  the  serum  of  such  animals  in  the  test-tube  is  capable  of 
destroying  the  corresponding  organisms  in  large  numbers,  and  that 
the  same  can  occur  in  the  living  animal,  but  we  must  also  recognize 
the  fact  that  this  flood  of  bacteriolysins  does  not  remain  while  the 
increased  resistance  that  has  been  established  may  last  for  years. 
That  the  phagocytic  influences  in  such  cases  do  not  play  a  more 
important  part  than  the  bacteriolysins,  can  readily  be  shown  by 
studying  the  opsonic  curve,  which  never  remains  above  the  normal 


138  THE  DIFFERENT  TYPES  OF  IMMUNITY 

for  any  length  of  time  after  the  infection  has  come  to  an  end,  if 
indeed  it  has  been  increased  at  any  time  during  its  course,  or  there- 
after. Evidently,  then,  still  other  influences  must  here  be  operative, 
but  what  these  influences  are  is  still  a  matter  of  speculation.  If  we 
bear  in  mind  that  a  cell  which  has  once  been  stimulated  to  active 
antibody  formation,  probably  responds  to  subsequent  stimuli  of 
the  same  order  with  greater  rapidity,  and  that  those  receptors  no 
doubt  are  regenerated  in  greatest  number  which  have  the  greatest 
affinity  for  the  particular  antigen  to  which  they  are  "tuned,"  we 
can  imagine  that  the  introduction  of  the  corresponding  organisms 
at  any  period  following  the  original  infection  or  vaccination  will 
be  successfully  overcome  in  consequence  of  this  specially  active 
response. 

This,  however,  is  as  yet  a  mere  supposition,  and  the  question 
still  remains  unanswered  why  infection  with  certain  organisms  leads 
to  immunity  and  not  with  others.  A  discussion  of  the  many  possi- 
bilities which  present  themselves  in  connection  with  this  problem 
would  serve  no  useful  purpose  at  this  place.  Much  work  still  remains 
to  be  done,  but  the  main  avenues  along  which  profitable  research 
should  be  conducted  are  already  clearly  indicated. 

Mechanism  of  Antitoxic  Immunity. — While  our  knowledge  of  the 
mechanism  underlying  the  development  of  antibacterial  immunity 
is  thus  still  very  fragmentary,  and  really  permits  a  clearer  insight 
into  the  manner  in  which  infection  can  take  place  than  into  the 
reasons  why  immunity  may  or  may  not  develop,  we  have  a  much 
better  understanding  of  the  modus  operandi  which  forms  the  basis 
of  the  antitoxic  type  of  immunity.  The  organisms  which  are  char- 
acterized by  a  high  degree  of  toxicity,  such  as  the  tetanus  and  the 
diphtheria  bacillus,  as  we  have  repeatedly  pointed  out,  possess  a 
very  low  grade  of  infectiousness,  so  that  they  readily  succumb  to 
the  normal  bactericidal  agencies  of  the  body.  Their  toxicity,  how- 
ever, is  of  such  a  high  order  that  they  are  nevertheless  formidable 
pathogenic  agents.  It  is  accordingly  surprising  to  find  that  some 
animals  are  absolutely  immune  to  the  action  of  these  toxins,  and, 
as  a  matter  of  principle,  it  is  important  to  learn  to  what  agencies 
this  remarkable  natural  immunity  is  due.  In  this  connection 
Ehrlich's  side-chain  theory  regarding  the  origin  and  formation  of 
antibodies  has  been  very  helpful  in  arriving  at  a  fairly  definite 
understanding. 


MECHANISM  OF  ANTITOXIC  IMMUNITY  139 

Different  possibilities,  of  course,  suggest  themselves.  Since  a  toxic 
effect  presupposes  the  existence  on  the  part  of  some  of  the  body 
cells  of  special  molecular  groups  with  which  the  toxins  can  combine, 
it  stands  to  reason  that  a  natural  absence  of  such  groups  must  lead 
to  natural  immunity  so  far  as  that  special  toxin  is  concerned.  But 
if  this  be  the  case  then  the  formation  of  a  corresponding  antitoxin 
should  not  be  possible.  By  this  criterion,  then,  we  can  test  any  cases 
that  might  suggest  themselves  as  belonging  to  this  order.  Metsch- 
nikoff  has  pointed  out  that  certain  reptiles,  and  notably  the  turtle, 
are  naturally  absolutely  immune  to  tetanus  toxin;  no  matter  whether 
the  animals  be  kept  at  the  ordinary  temperature  of  the  aquarium, 
or  at  37°  C.,  following  an  injection  of  the  toxin,  their  blood  remains 
highly  toxic  for  mice,  even  for  several  months.  Coincidently  he 
found  that  there  was  not  the  slightest  formation  of  antitoxin.  This 
example  then  illustrates  especially  well  the  actual  existence  of  a 
theoretically  possible  form  of  immunity  due  to  absence  of  suitable 
receptors. 

A  second  possibility  suggests  itself  if  we  bear  in  mind  that  not 
all  cells  which  may  possess  suitable  combining  groups  for  a  toxin 
molecule  are  necessarily  deleteriously  affected  by  such  a  union. 
In  such  an  event  we  should  expect  absence  of  toxic  effect  associated 
with  the  production  of  antitoxin,  for  we  have  seen  that  the  latter 
can  take  place  perfectly  well  even  though  the  specific  action  of  the 
toxophoric  group  is  eliminated.  That  this  may  actually  occur  in 
nature  is  well  shown  in  the  case  of  the  American  alligator,  which 
is  as  resistant  to  the  action  of  the  tetanus  toxin  as  is  the  turtle, 
but  which,  unlike  the  latter,  furnishes  an  abundant  amount  of 
corresponding  antitoxin.  That  the  toxin  in  this  case  is  actually 
bound  by  the  cells  is  also  shown  by  the  fact  that,  contrary  to  what 
we  have  noted  in  the  turtle,  it  rapidly  disappears  from  the  circu- 
lation. In  such  a  case  the  immunity  is  evidently  not  due  to  absence 
of  suitable  receptors,  but  to  an  insusceptibility  on  the  part  of  the 
binding  cells. 

Still  another  possibility  would  exist,  if  both  susceptible  and 
insusceptible  cells  were  present  in  the  body,  but  if  the  latter  possessed 
a  greater  affinity  for  the  toxin  than  the  former.  In  such  a  case  we 
should  theoretically  expect  active  antitoxin  formation,  immunity 
to  small  doses  of  the  toxin,  but  absence  of  immunity  to  a  larger 
dose,  the  result,  moreover,  varying  with  the  point  at  which  the 


140  THE  DIFFERENT  TYPES  OF  IMMUNITY 

toxin  is  introduced.  If  this  should  occur  in  a  territory  which  con- 
tains large  numbers  of  insusceptible  cells  (though  provided  with 
suitable  haptophoric  groups)  no  deleterious  results  would  be  expected, 
while  in  the  opposite  case  the  consequences  would  of  necessity  be 
disastrous.  A  great  deal,  moreover,  other  things  being  equal,  would 
depend  upon  the  size  of  the  dose,  for  if  this  should  exceed  the  demands 
of  the  insusceptible  cells  a  toxic  effect  would  naturally  be  the  out- 
come. In  such  instances,  then,  the  immunity  would  only  be  relative. 
This  is  exactly  what  we  see  in  the  case  of  the  rabbit,  which  is  rela- 
tively insusceptible  to  tetanus  toxin,  when  this  is  administered 
hypodermically,  but  highly  sensitive  if  the  poison  is  injected  directly 
into  the  brain. 

It  will  be  noted  that  these  three  different  types  of  immunity  are 
thus  essentially  dependent  upon  the  character  of  the  cells,  i.  e., 
that  they  are  histogenetic  in  character,  and  that  the  examples  which 
have  served  as  illustrations  at  the  same  time  represent  types  of 
natural  immunity.  But  we  have  also  seen '  that  immunity  (sc., 
increased  resistance)  must  result  if  for  any  reason  antitoxin  mole- 
cules enter  the  circulation  in  sufficient  number  to  neutralize  any 
toxin  that  may  be  present.  Immunity  of  this  order  is  thus  humoral 
in  character  and  usually,  if  not  always,  acquired.  This  may  result 
as  a  consequence  of  infection  or  immunization,  and  then  represents 
a  type  of  active  immunity,  or  it  is  acquired  in  a  passive  manner, 
the  organism  of  the  individual  taking  no  part  in  its  production 
(passive  immunity) .  An  example  of  the  first  type  is  furnished  by  the 
antitoxin  horses,  in  which  a  high  degree  of  immunity  is  produced 
by  systematic  immunization,  while  the  production  of  passive  immu- 
nity is  illustrated  in  the  prophylactic  treatment  of  diphtheria  or 
tetanus  with  the  corresponding  antitoxic  sera.  To  the  latter  order 
also  belongs  the  immunity  which  is  conveyed  by  actively  immunized 
animals  to  their  offspring,  either  during  intra-uterine  life  or  post 
partum  through  the  milk. 

While  the  underlying  principle  of  these  types  of  immunity  is  thus 
quite  well  understood,  still  another  form  of  acquired  immunity  is, 
theoretically  at  least,  possible.  We  have  seen  that  under  natural 
conditions  a  form  of  antitoxin  immunity  exists,  which  is  referable 
to  absence  of  suitable  haptophoric  groups  on  the  part  of  the  body 
cells.  Theoretically  it  is  conceivable  that  such  a  form  of  immunity 
might  also  be  acquired,  if  in  any  way  atrophy  of  the  corresponding 


MECHANISM  OF  ANTITOXIC  IMMUNITY  141 

receptors  were  to  occur  either  in  all  cells,  or  only  in  those  cells  which 
are  susceptible  to  toxin  influence.  As  a  matter  of  fact,  there  is 
experimental  evidence  to  show  that  this  may  occur.  Thus,  while 
the  red  blood  corpuscles  of  the  normal  rabbit  are  readily  destroyed 
by  the  peculiar  toxin  which  is  found  in  the  serum  of  eels,  the  cor- 
puscles of  correspondingly  immunized  animals,  even  though  washed 
free  from  any  antitoxin  that  may  be  present  in  the  serum,  are  abso- 
lutely resistant.  Evidently  they  have  lost  the  receptors  which  in 
the  non-immune  animal  made  the  action  of  the  toxin  possible.  It 
has  similarly  been  observed  that  animals  which  have  been  highly 
immunized  against  diphtheria  toxin  may  finally  cease  the  produc- 
tion of  antitoxin  altogether,  and  simultaneously  lose  their  suscep- 
tibility to  the  toxin  in  question  altogether,  phenomena  which  are 
most  readily  explained  upon  the  basis  of  acquired  atrophy  of  the 
corresponding  receptors. 

While  these  examples  plainly  illustrate  the  undoubted  occurrence 
of  an  acquired  antitoxic  immunity,  due  to  receptoric  atrophy,  there 
are  further  observations  which  show  that  this  principle  also  plays 
an  important  role  in  the  development  of  other  types  of  immunity. 
Thus  far  we  have  only  considered  the  reaction  of  the  macro  organism 
to  the  introduction  of  the  microorganism,  and  the  question  very 
naturally  suggests  itself,  Is  it  not  possible  that  the  microorganism 
may  become  resistant  to  the  deleterious  influences  which  it  meets 
within  its  host?  We  have  seen  that  it  may  protect  itself  by  the 
development  of  capsules  and  the  liberation  of  aggressins.  Within 
recent  years,  observations  have  come  to  light,  however,  which  make 
it  very  probable  that  the  principle  of  receptoric  atrophy  may  play 
an  important  role  here  also.  Much  of  this  work  and  its  brilliant 
interpretation  we  likewise  owe  to  the  genius  of  Ehrlich.  He  has 
shown  that  on  treating  rats  which  have  been  infected  with  trypano- 
somes  (S  I)  with  an  amount  of  arsenophenyl  glycin,  arsanil,  or 
arsacetin,  not  quite  sufficient  to  kill  all  the  organisms,  trypanocidal 
antibodies  are  produced,  which  Ehrlich  conceives  to  be  the  outcome 
of  the  antigenic  effect  of  the  ordinary  nutriceptors1  of  the  parasite, 
upon  those  cells  of  the  macroorganisms  which  are  provided  with  cor- 
responding haptophoric  groups.  Those  trypanosomes  which  have  not 
been  killed  by  the  arsenic  now  find  themselves  in  the  presence  of 

1  Nutriceptors  are  here  understood  to  be  those  receptors  which  serve  the 
nutrition  of  the  organism  (sc.,  the  cell). 


142  THE  DIFFERENT  TYPES  OF  IMMUNITY 

these  antibodies  (A  I),  and  insofar  as  they  are  not  destroyed,  they 
respond  to  the  occupation  of  their  original  nutriceptors  (NT)  by  these 
antibodies  with  the  production  of  a  new  type  of  nutriceptors  (NTT), 
which  we  may  imagine  to  possess  a  greater  affinity  for  the  available 
foodstuffs  than  for  the  antibodies  that  are  simultaneously  present. 
A  new  strain  of  trypanosomes  thus  develops  in  which  this  peculiarity 
is  handed  down  from  each  individual  parasite  to  its  descendants.  If 
this  strain  (S  II)  is  now  tested  against  a  serum  containing  antibodies 
of  the  type  A  I,  it  will  be  found  immune,  and  as  Ehrlich  has  pointed 
out,  this  type  of  immunity  can  be  explained  only  in  the  manner 
just  outlined,  viz.,  on  the  basis  of  receptoric  atrophy. 

The  importance  of  this  principle  in  the  interpretation  of  various 
phases  of  human  and  animal  pathology  is,  of  course,  evident.  It 
readily  explains,  for  example,  why  the  syphilitic  individual  is  refrac- 
tory to  reinoculation,  while  he  is  liable  to  relapses  starting  from  his 
original  infection.  We  may  imagine  that  in  such  a  person,  different 
strains  of  spirochetes  develop  as  a  result  of  adaptation  to  those 
antibodies  which  are  formed  in  consequence  of  the  death  and  absorp- 
tion of  the  first  and  subsequently  developing  strains,  and  that  the 
latest  strain,  in  point  of  time  of  development,  will  always  be  capable 
of  causing  a  relapse,  as  no  suitable  antibodies  to  it  have  as  yet 
developed  and  because  it  is  immune  to  those  that  have  been  formed 
before.  The  number  of  strains  which  can  theoretically  be  produced 
in  the  course  of  an  infection  will,  no  doubt,  vary  with  different 
organisms,  as  well  as  with  the  nature  of  the  host.  A  great  deal  of 
additional  work  will  have  to  be  done,  however,  before  we  can  speak 
with  any  degree  of  definiteness  on  this  subject.  Ehrlich  has  shown 
that  in  the  case  of  the  spirillum  of  relapsing  fever  only  three  or  four 
strains  are  possible.  If,  then,  the  patient  or  animal  has  had  two  or 
three  relapses  the  body  will  contain  all  the  different  "strains"  of 
spirillocidal  antibodies  that  are  possible,  no  new  strain  can  accord- 
ingly develop,  and  spontaneous  recovery  will  occur.  The  greater 
the  number  of  strains  which  can  develop  the  greater  will  naturally 
be  the  obstacles  to  spontaneous  recovery.  This  holds  good  espe- 
cially for  such  diseases  as  syphilis  and  trypanosomiasis  (sleeping 
sickness),  and  possibly  also  for  malaria. 

From  these  brief  considerations  it  will  be  seen  that  the  subject 
of  immunity  referable  to  receptoric  atrophy  is  a  most  important 
one,  and  that  we  may  reasonably  expect  much  valuable  information 


MECHANISM  OF  ANTITOXIC  IMMUNITY  143 

from  a  continued  and  more  detailed  investigation  of  the  subject. 
Since  the  same  principle,  moreover,  seems  to  apply  not  only  to  immu- 
nity to  infection,  but  also  to  the  question  of  resistance  to  various 
chemical  agents  on  the  part  of  various  low  forms  of  animal  and 
vegetable  life,  it  is  clear  that  the  subject  must  also  be  of  great 
interest  from  the  standpoint  of  therapeutics,  and  furnishes  a  logical 
basis  for  the  now  generally  recognized  fact,  that  in  the  medicinal 
treatment  of  certain  infections,  like  syphilis,  our  aim  should  be  a 
therapia  magna  sterilisans,  rather  than  the  continued  administration 
of  small  doses  of  certain  drugs  (see  section  on  Chemotherapy). 

In  fine  we  may  say  that  much  has  already  been  learned  of  the 
manner  in  which  immunity  may  develop,  but  much  more  still 
remains  to  be  known.  The  avenues  along  which  further  investi- 
gations may  be  profitably  pursued  are  already  well  defined  and  we 
may  confidently  expect  much  valuable  new  information  in  the  near 
future. 


CHAPTER  X 
ANAPHYLAXIS 

IT  has  long  been  recognized  that  while  certain  infections,  such 
as  smallpox,  scarlatina,  measles,  whooping  cough,  typhoid  fever, 
cholera,  typhus  fever,  etc.,  lead  to  immunity,  others  not  only  bring 
about  no  increased,  but  actually  a  decreased  resistance  to  subsequent 
infection  with  the  same  organism.  This  is  notably  true  of  pneumonia, 
erysipelas,  influenza,  diphtheria,  bacillary  dysentery,  certain  staphy- 
lococcus  infections,  such  as  tonsillitis,  acne,  etc.  In  the  past  we 
have  been  totally  unable  to  explain  these  peculiar  differences,  and 
even  now  our  knowledge  of  the  mechanism  underlying  the  production 
of  hypersusceptibility  to  certain  deleterious  influences  is  very  meager. 
Within  recent  years,  however,  such  a  wealth  of  experimental  facts 
has  been  accumulated  which  have  a  direct  bearing  upon  the  problem 
under  consideration,  that  the  day  no  longer  seems  far  distant  when 
we  shall  be  able  to  offer  an  adequate  explanation  for  these  peculiar 
differences.  Some  of  these  observations  and  the  resulting  deduc- 
tions will  be  considered  in  the  present  chapter. 

In  the  foregoing  pages  we  have  explained  the  manner  in  which 
immunity  to  toxins  may  be  artificially  brought  about,  and  have 
shown  that  this  depends  essentially  upon  the  liberation  of  corre- 
sponding receptors  on  the  part  of  some  of  the  body  cells.  If  these 
are  produced  and  thrown  off  in  sufficiently  large  number,  they  fur- 
nish a  protection  for  the  body  against  the  toxins  in  question  which 
may  be  of  a  very  high  order.  In  the  commercial  preparation  of 
antitoxins  it  is,  of  course,  desirable  to  obtain  sera  that  shall  be  as 
potent  as  possible,  and  it  is  hence  customary  to  force  the  process 
of  immunization  in  the  experimental  animals  to  the  highest  limit. 
The  question  now  arises  what  happens  if  this  be  exceeded.  Two 
events  may  then  occur.  On  the  one  hand  the  animal  may  cease  to 
produce  antitoxin  altogether,  but  simultaneously  loses  all  suscepti- 
bility to  the  corresponding  toxin,  and  is  thus  absolutely  immune. 

This  result,  as  we  have  already  shown,  is  explained  on  the  basis  of 
an  acquired  receptoric  atrophy,  and  we  can  readily  conceive  that  this 


ANAPHYLAXIS  145 

should  occur,  if  the  specific  receptors  which  the  cell  forms  under 
the  stimulus  of  the  toxin  are  continuously  cast  off,  and  thus  no  longer 
serve  a  useful  purpose  so  far  as  the  nutrition  of  the  cell  is  concerned. 
On  the  other  hand,  the  opposite  may  occur.  The  animal,  while 
actively  forming  antitoxin,  loses  its  increased  resistance  to  the  corre- 
sponding toxin,  and  succumbs  to  a  much  smaller  dose  of  the  latter 
than  the  original  minimal  fatal  dose.  It  is  thus  no  longer  immune, 
but  actually  hypersensitive.  As  this  phenomenon  is  only  observed 
in  actively  and  never  in  passively  immunized  animals,  the  conclusion 
suggests  itself  that  its  basis  must  be  histogenetic  and  not  humoral 
in  character.  Since  antitoxin  is  present  in  the  blood  of  the  animals 
in  large  amount  we  must  suppose  that  this  actually  anchors  the  toxin, 
but  as  the  animal  dies  with  typical  toxin  symptoms  we  must  also 
conclude  that  the  toxin-antitoxin  combination  is  again  severed  and 
that  the  toxin  after  all  reaches  the  corresponding  receptors  of  the 
susceptible  cells.  This,  of  course,  would  presuppose  the  existence 
of  a  higher  affinity  for  the  toxin  on  the  part  of  the  sessile  than  of 
the  circulating  receptors.  That  there  is  actually  a  basis  for  such 
an  assumption  has  been  shown  by  Miiller,  who  could  demonstrate 
that  at  any  one  time  the  blood  serum  of  an  animal  undergoing 
immunization  contains  antibodies  of  varying  degrees  of  affinity  for 
the  corresponding  antigen,  and  that  those  possessing  the  highest 
affinity  are  the  latest  formed. 

The  hypersusceptibility  of  the  highly  immunized  animal  would 
thus  find  a  ready  explanation,  which  would  also  seem  to  apply  in 
the  case  of  the  so-called  paradox  of  Kretz.  This  investigator  found 
that  while  the  injection  of  an  accurately  neutralized  toxin-antitoxin 
mixture  produces  no  deleterious  results  whatever  in  the  normal 
animal,  in  one  which  has  been  previously  actively  immunized  with 
toxin  the  reverse  occurs.  Here  also  we  may  suppose  that  as  the 
result  of  the  immunization,  highly  active  receptors  are  present 
in  the  susceptible  cells,  and  that  these  are  capable  of  displacing 
the  antitoxin  which  has  been  added  in  vitro  and  of  anchoring  the 
liberated  toxin  which  then  acts  upon  the  cell  before  this  has  cast 
off  the  corresponding  receptors. 

If  coincidently  in  either  one  of  the  two  instances  just  considered 
receptoric  atrophy  should  develop  in  the  non-susceptible  cells  it  is 
clear  that  the  susceptible  cells  would  become  even  more  liable  to 
attack  by  the  toxin. 
10 


146  ANAPHYLAXIS 

Probably  belonging  to  the  same  order  of  cellular  hypersuscepti- 
bility  is  also  the  increased  susceptibility  of  the  tubercular  organism 
to  the  introduction  of  tuberculin  in  doses  which  in  the  normal 
individual  produce  no  reaction  whatever.  We  may  here  imagine 
that  in  tubercular  foci,  sessile  receptors  are  present  in  large  numbers 
which  possess  a  greater  affinity  for  the  tubercular  antigen  (tuber- 
culin) than  do  the  receptors  of  any  normal  cells,  and  that  these 
receptors  eagerly  take  up  the  corresponding  antigen,  when  this  is 
introduced  from  without.  The  specific  reaction  which  then  takes 
place  we  can  conceive  to  be  due  to  an  interaction  between  antigen 
(tuberculin)  and  antibody  (receptor),  with  the  consequent  produc- 
tion of  toxic  products  and  their  action  upon  the  cells  in  question. 
This  view  is  supported  by  the  discovery  on  the  part  of  Wassermann 
and  Bruck  that  tubercular  organs  actually  contain  specific  sub- 
stances which  will  combine  with  tubercular  antigen,  as  can  be 
demonstrated  with  the  complement  fixation  method  (which  see). 

Richet's  Early  Investigations. — A  marked  impetus  to  the  study  of 
hypersusceptibility  was  then  given  by  certain  observations  of  Richet 
(1902).  This  investigator  found  that  the  intravenous  injection  into 
dogs  of  extracts  made  from  the  tentacles  of  certain  actiniae  pro- 
duced marked  toxic  symptoms  (excitement,  bloody  diarrhea,  and 
subnormal  temperature),  which  appear  after  a  certain  interval,  then 
increase  in  severity  during  the  first  two  days,  and  lead  to  a  fatal 
issue  only  at  the  expiration  of  the  third  day.  Post  mortem  he  found 
marked  congestion  of  the  viscera  (stomach,  intestines,  liver,  and 
kidneys),  and  he  accordingly  termed  the  toxic  principle  in  question 
actinocongestin.  He  further  ascertained  that  very  curiously  the 
immediate  repetition  of  a  fatal  dose  of  the  poison  never  produced 
sudden  death,  but  that  the  end  was  invariably  delayed  until  the 
expiration  of  the  third  day.  If  now  an  animal  is  injected  with  a 
non-fatal  dose  of  the  poison,  and  after  recovery  from  its  effects  is 
reinjected  with  an  amount  which  in  an  animal  that  had  previously 
not  been  injected  would  produce  no  deleterious  effects  whatever 
(such  as  one-twentieth  of  the  original  quantity),  most  serious 
symptoms  develop  at  once  and  the  animal  dies  within  twelve  to 
twenty-four  hours.  In  a  concrete  case  0.08  gram  was  used  in  the 
first  injection  without  producing  any  vomiting,  while  a  reinjection 
of  only  0.001  gram  gave  rise  to  this  at  once.  In  other  words,  at  the 
time  of  the  second  injection  the  animal  was  eighty  times  more 


EARLY  STUDIES  OF  V.  PIRQUET  147 

sensitive  than  before  the  first.  Richet  further  showed  that  while 
the  primary  injection  produces  no  material  effect  upon  the  blood 
pressure  the  second  injection  is  followed  by  a  marked  drop. 

Evidently  then  the  first  injection  has  in  some  manner  called  forth 
a  hypersusceptibility  to  the  special  toxin,  which  in  the  present 
instance  is  characterized  by  an  increased  velocity  as  well  as  an 
increased  intensity  of  reaction.  This  type  of  hypersensitiveness, 
Richet  has  termed  anaphylaxis,  indicating  the  absence  of  protection, 
in  contradistinction  to  prophylaxis  or  immunity. 

Arthus  Phenomenon. — The  following  year  (1903)  Arthus  then 
showed  that  similar  results  may  be  obtained  with  substances  which 
unlike  the  actinocongestin  are  altogether  non-toxic.  For  on  inject- 
ing rabbits  at  definite  intervals  with  normal  horse  serum,  he  found 
that  the  first  two  or  three  doses  were  promptly  absorbed,  but  that 
subsequent  injections  led  to  increasingly  more  severe  local  reactions, 
so  that  at  times  gangrene  even  developed.  This  occurred  no  matter 
whether  the  injections  were  all  made  subcutaneously,  or  the  first  ones 
given  intraperitoneally  and  only  the  last  ones  hypodermically.  If 
the  animals,  moreover,  were  first  injected  subcutaneously,  and  subse- 
quently intraperitoneally  or  intravenously,  serious  general  disturb- 
ances (dyspnea,  diarrhea,  convulsions)  and  even  death  resulted 
(Arthus  phenomenon).  Corresponding  results  were  obtained  with 
milk,  and  Arthus  could  show  that  the  anaphylactic  reaction  in  ques- 
tion was  specific,  as  an  animal  that  had  been  sensitized  with  horse 
serum,  for  example,  was  not  injured  by  the  subsequent  injection 
of  either  milk,  white  of  egg,  or  the  serum  of  other  animals,  but 
only  of  horse  serum. 

Early  Studies  of  v.  Pirquet. — Corresponding  clinical  studies  were 
undertaken  almost  simultaneously  by  v.  Pirquet,  and  were  based 
upon  the  independent  observation  that  a  second  injection  of  horse 
serum  in  a  child  was  not  followed  by  symptoms  of  serum  sickness 
at  the  expiration  of  ten  days,  as  had  been  noted  after  the  first  injec- 
tion, but  that  they  occurred  in  the  course  of  the  same  day  on  which 
the  second  injection  was  given.  He  concluded  that  the  then  existing 
doctrine  regarding  the  time  of  incubation  in  the  different  infectious 
diseases  was  erroneous,  and  propounded  the  hypothesis  that  the 
pathogenic  agent  calls  forth  symptoms  of  disease  only  after  it  has 
been  changed  by  corresponding  antibodies,  and  that  the  period 
of  incubation  represents  the  interval  of  time  which  is  necessary  for 


148  ANAPHYLAXIS 

antibody  formation.  Subsequently  he  showed  in  a  joint  publication 
with  Schick  that  his  original  observation  merely  illustrated  the 
general  rule  that  a  first  injection  of  horse  serum  always  sensitizes 
the  individual  to  subsequent  injections,  so  that  the  latter  are  followed 
by  symptoms  more  rapidly  and  more  uniformly  and  can  be  produced 
by  doses  which  are  much  smaller  than  the  first  ones. 

In  contradistinction  to  Arthus  who  ascribed  the  hypersensitive- 
ness  of  his  animals  to  the  repetition  of  the  injections  in  a  general 
way,  and  who  thought  that  it  increased  in  intensity  with  each 
injection,  v.  Pirquet  and  Schick  emphasized  that  a  single  injection 
suffices  to  bring  about  this  result,  and  that  a  certain  interval  must 
elapse  before  the  animal  responds  in  the  changed  manner  to  the 
second  injection.  If,  for  example,  the  injection  is  repeated  after 
five  days,  no  induration  develops  at  the  site  of  the  puncture,  while 
at  the  expiration  of  ten  days  this  is  very  marked.  Subsequent 
injections  usually  lead  to  still  more  marked  reactions,  but  v.  Pirquet 
has  shown  that  even  then  a  diminution  in  susceptibility  may  occur, 
and  that  hypersensibility  and  immunity  can  accordingly  not  be 
separated  in  principle. 

Theobald  Smith  Phenomenon. — Further  experimental  studies  were 
then  called  forth  by  the  observation  of  Theobald  Smith  that  guinea- 
pigs  which  had  once  been  used  in  the  titration  of  diphtheria  anti- 
toxin and  which  had  hence  been  injected  with  a  toxin-antitoxin 
mixture  were  thereafter  hypersensitive  to  subsequent  injections  of 
horse  serum.  If  such  animals  were  reinjected  they  showed  immediate 
symptoms  of  a  serious  character;  they  became  restless,  dyspneic,  the 
heart  action  became  feebler  and  feebler,  the  temperature  dropped 
below  the  normal,  and  in  fully  50  per  cent,  death  occurred  within 
a  half  hour  (Theobald  Smith  phenomenon).  Post  mortem,  a  most 
striking  picture  was  seen  which  readily  explained  the  majority  of  the 
symptoms  which  preceded  the  fatal  end,  for  on  widely  opening  the 
thorax  the  lungs  did  not  collapse,  but  remained  rigid  in  a  state  of 
deepest  inspiration.  This  phenomenon  was  first  described  by  Auer 
and  Lewis,  and  is  attributed  by  these  investigators  to  spasm  of 
the  smallest  bronchioles,  which  virtually  causes  the  suffocation  of 
the  animal. 

At  Ehrlich's  suggestion,  his  pupil  Otto  took  up  the  investigation 
of  this  problem  and  almost  simultaneously  with  his  report  there 
appeared  a  detailed  study  of  the  same  subject  by  Rosenau  and 


ANTIANAPHYLAXIS  149 

Anderson.  From  the  experiments  of  these  observers  it  appears  that 
the  toxin  in  itself  has  nothing  to  do  with  the  sensitization  of  the 
animals,  and  that  the  horse  serum  produces  this  effect  only  if  used 
in  small  doses,  but  that  the  toxin  in  some  manner  which  is  not  yet 
understood  facilitates  the  sensitizing  influence  of  the  serum.  The 
small  size  of  the  dose  of  serum  which  in  itself  is  sufficient  to  cause 
sensitization  is  indeed  remarkable.  In  one  instance  Rosenau  and 
Anderson  produced  this  result  with  yinmnnr  °f  a  c-c->  while 
amounts  ranging  from  2"i~o  to  yoVo"  c-c-  were  sufficient  in  every 
instance;  and  while  a  first  injection  of  10  c.c.,  which  is  equivalent  to 
40  c.c.  pro  kilo  of  animal,  caused  no  symptoms  of  any  kind  in  the 
guinea-pig,  0.1  c.c.  as  second  injection  was  sufficient  to  cause 
death. 

Like  Arthus  they  found  the  reaction  to  be  specific  insofar  as  it 
was  impossible  to  produce  symptoms  in  animals  that  had  been 
sensitized  with  horse  serum,  by  subsequently  injecting  them  with  the 
serum  from  animals  of  a  different  species.  Like  v.  Pirquet,  they 
could  also  show  that  a  certain  time  interval  must  elapse  between 
the  two  injections  before  anaphylactic  symptoms  develop,  and  that 
this  depends  to  a  certain  extent  upon  the  point  at  which  the  first 
injection  is  given;  if  this  is  made  into  the  brain  the  animal  becomes 
sensitive  after  the  eighth  day,  while  following  a  subcutaneous  injec- 
tion this  occurs  at  least  two  days  later. 

Antianaphylaxis. — Especially  interesting  also  was  the  discovery 
that  if  an  animal  is  reinjected  shortly  before  the  twelfth  day  the 
reaction  is  only  slight  or  may  not  occur  at  all,  and  that  subsequent 
injections,  for  a  certain  time  at  least,  produce  no  deleterious  conse- 
quences; in  other  words,  the  animal  has  become  resistant  instead 
of  hypersensitive  (antianaphylaxis) .  This  condition,  however,  is 
not  permanent  and  after  a  number  of  weeks  the  animals  gradually 
become  hypersensitive  again.  If,  however,  the  injections  are 
repeated  in  increasing  doses  and  properly  spaced  a  state  of  resist- 
ance can  be  produced  which  lasts  for  many  months. 

The  same  condition  may,  however,  also  be  brought  about  at  a 
time  when  the  animal  is  already  hypersensitive,  by  injecting  it  with 
a  sublethal  dose  of  the  antigen.  The  animal  may  then  become 
violently  ill,  it  is  true,  but  it  will  recover,  and  is  then  markedly 
antianaphylactic,  so  that  it  can  be  injected  even  after  a  very 
brief  interval  with  possibly  a  hundred  fold  larger  dose  of  the 


150  ANAPHYLAXIS 

antigen  without  any  deleterious  results  (see  also  Mechanism  of 
Antianaphylaxis) . 

Anaphylactogens. — Subsequent  investigations  have  then  shown 
that  an  anaphylactic  reaction  can  be  called  forth  by  the  injection 
not  only  of  blood  serum,  but  also  of  milk,  albuminous  urine,  sweat, 
bile,  red  cells,  extracts  of  various  normal  tissues  as  well  as  of  neo- 
plasms, the  contents  of  echinococcus  cysts,  extracts  of  lower  animals 
or  of  vegetable  organisms,  including  bacteria,  etc.,  in  short  by  any 
substance  of  albuminous  character,  and  it  is  especially  noteworthy 
that  this  in  itself  need  not  be  toxic  to  the  slightest  degree.  It  seems, 
indeed,  as  though  true  toxins  could  not  produce  anaphylaxis,  and 
that  if  this  apparently  occurs,  it  is  due  to  contaminating  albuminous 
substances.  This,  however,  does  not  preclude  the  possibility  that 
toxic  albumins  may  give  rise  to  the  reaction,  and  we  have  seen, 
as  a  matter  of  fact,  that  Richet's  original  experiments  were  carried 
out  with  such  material.  In  such  an  event,  of  course,  the  toxic 
character  of  the  albumins  may  blur  the  picture  somewhat;  this  is 
what  actually  occurred  in  the  case  of  Richet's  actinocongestin, 
and  no  doubt  led  to  his  assumption  that  the  extract  contained 
both  a  toxic  (anaphylactic)  substance  and  a  non-toxic  immunizing 
(prophylactic)  principle. 

Collectively  those  substances  which  are  capable  of  rendering  an 
animal  anaphylactic  are  spoken  of  as  anaphylactogens,  allergens,  or 
sensibilisinogens. 

Serum  Sickness. — It  was  next  shown  that  any  animal  may  be 
rendered  anaphylactic,  but  that  the  mode  and  intensity  of  the 
reaction  is  not  the  same  in  all.  The  most  susceptible  animal  is 
evidently  the  guinea-pig,  and  we  have  already  seen  the  manner  in 
which  it  reacts  to  the  introduction  of  horse  serum.  In  man  the 
same  antigen  leads  to  those  symptoms  which  collectively  are  spoken 
of  as  serum  sickness,  the  most  common  of  which  are  the  occurrence 
of  fever,  of  exanthemata,  and  of  swelling  of  the  joints.  In  dogs  we 
note  great  restlessness,  crying  out  aloud,  and  marked  fall  in  blood- 
pressure,  non-coagulability  of  the  blood,  and  leukopenia.  In  goats 
extreme  myosis  has  been  observed. 

Passive  Anaphylaxis. — Most  important  further  is  the  observation 
that  the  anaphylactic  reaction  product  (the  anaphylactin  or  sensi- 
bilisin  of  the  French;  the  oiler  gin  of  v.  Pirquet)  can  be  transferred 
from  one  animal  to  another,  in  a  manner  quite  analogous  to  the 


PRODUCTION  OF  THE  ANAPHYLACTIC  SHOCK  151 

production  of  passive  immunity,  and  writers  hence  speak  of  passive 
anaphylaxis ,  which  may  be  homologous  or  heterologous,  i.  e.,  it 
can  be  transferred  to  an  animal  of  the  same  species  or  to  one  of  a 
species  which  is  different  from  the  one  which  was  actively  sensitized. 
The  discovery  of  this  fact  has  had  important  bearings  upon  our 
understanding  of  the  mechanism  which  underlies  the  production 
of  the  anaphylactic  shock,  for  it  showed  conclusively  that  humoral 
factors  are  here  at  play. 

Production  of  the  Anaphylactic  Shock. — Richet  originally  propounded 
the  hypothesis  that  as  a  result  of  the  first  injection  a  special  anti- 
body is  formed,  which  he  termed  toxogenin,  and  that  this  then  splits 
off  a  highly  toxic  poison  from  the  primary  toxin,  for  example, 
from  the  anaphylactogenic  principle  of  his  actinocongestin. 
This  hypothesis,  however,  cannot  be  applied  to  the  anaphylactic 
reaction  which  follows  the  administration  of  non-toxic  antigens, 
and  is  evidently  based  upon  false  premises.  Other  writers,  such 
as  Weichardt,  v.  Pirquet  and  Schick,  Wolff-Eisner,  Friedberger, 
Friedemann  and  Isaac,  also  assume  the  formation  of  antibodies, 
but  suppose  that  a  special  toxin  is  set  free  from  the  corresponding 
non-toxic  antigen  when  the  two  meet.  Regarding  the  manner  in 
which  this  occurs,  different  possibilities,  of  course,  suggest  them- 
selves. Led  by  his  observations  on  the  anaphylactic  reaction  which 
follows  the  introduction  of  alien  cells  into  the  rabbit,  Wolff-Eisner 
assumed  a  lytic  action  on  the  part  of  the  anaphylactic  antibody 
upon  the  corresponding  albuminous  antigen,  analogous  to  the  lytic 
action  of  the  cytotoxins  (e.  g.,  bacteriolysins)  and  a  consequent 
liberation  of  endotoxin-like  substances.  Weichardt  arrived  at 
similar  conclusions  on  the  basis  of  analogous  experiments  with 
placental  cells,  but,  unlike  Wolff-Eisner,  he  assumed  that  the  lytic 
action  of  the  antibody  does  not  set  free  preformed  endotoxins,  but 
that  the  lysis  is  followed  by  further  chemical  changes. 

More  recent  investigations,  notably  by  Dorr  and  Russ,  have 
rendered  it  highly  probable  that  the  antibody  in  question  is  really 
a  precipitin,  and  the  predominating  idea  at  present  is  that  an  ana- 
phylactic toxin  is  in  some  manner  split  off  from  the  corresponding 
precipitate  through  the  agency  of  complement.  This  view  is,  as  a 
matter  of  fact,  supported  by  numerous  observations.  It  has  thus 
been  shown  that  those  split-products  of  the  albumins  which  no  longer 
give  rise  to  precipitin  formation,  likewise  do  not  act  as  sensibilisino- 


152  ANAPHYLAXIS 

gens,  and  that  there  does  not  exist  a  single  precipitinogenic  protein 
which  has  not  also  anaphylaxis-producing  properties.  Precipitating 
sera,  moreover,  always  contain  the  anaphylactic-reaction  product. 
Whether  or  not  special  albuminolytic  amboceptors  may  also  be 
concerned  in  the  anaphylactic  reaction  must  thus  remain  an  open 
question.  So  much  is  certain  that  precipitin  formation  and  anaphyl- 
actin  formation  evidently  run  a  parallel  course,  and  that  there  is 
no  good  reason  for  doubting  their  identity.  This,  at  least,  seems 
established  for  the  anaphylactic  reaction  product  which  is  formed 
as  the  result  of  the  parenteral  introduction  of  albumins.  In  the  case 
of  animal  or  vegetable  cells,  on  the  other  hand,  there  is  evidence 
to  show  that  the  cytolytic  amboceptors  may  play  the  role  of  the 
anaphylactins. 

Complement  and  Production  of  Anaphylactic  Toxin. — That  comple- 
ment is  necessary  for  the  production  of  the  anaphylactic  toxin  has 
been  demonstrated  beyond  a  doubt.  Friedemann  and  Friedberger 
have  thus  shown  that  when  fresh  complement  is  added  to  a  mixture 
of  an  albumin  and  its  corresponding  antiserum,  in  the  test-tube,  a 
toxic  product  (anaphylatoxin)  is  formed  which,  upon  injection  into 
a  suitable  animal,  calls  forth  practically  all  the  characteristic  symp- 
toms of  anaphylaxis.  Quite  in  accord  with  this  observation  is  the 
fact  that  during  the  anaphylactic  reaction,  produced  in  the  usual 
wray,  the  complement  of  the  blood  is  reduced  to  one-fifth  or  even 
to  one-half  of  the  original  amount,  and  that  the  shock  cannot  be 
prevented  by  the  artificial  introduction  from  without  of  comple- 
ment, even  in  large  amount.  Moreover,  if  an  animal  (such  as  the 
pigeon)  which  is  biologically  far  removed  from  the  rabbit,  and  w^hose 
complement  does  not  supplement  the  action  of  any  amboceptors 
formed  in  the  latter,  is  injected  with  the  serum  from  a  sensibilized 
animal  of  this  order,  and  then  reinjected  with  the  corresponding 
antigen,  no  anaphylactic  shock  should  theoretically  develop,  and, 
as  matter  of  fact,  does  not  develop.  As  in  the  test-tube  experiment, 
furthermore,  the  action  of  complement  can  be  prevented  through 
the  addition  of  a  suitable  amount  of  salt,  i.  e.,  by  raising  the  osmotic 
pressure  of  the  mixture;  so,  also,  is  it  possible  to  prevent  the  develop- 
ment of  the  anaphylactic  shock  in  sensitized  animals  by  a  preliminary 
injection  of  large  amounts  of  salt. 

Nature  of  Anaphylactic  Toxin. — Regarding  the  nature  of  the  ana- 
phylactic poison,  our  knowledge  is  as  yet  quite  meager.  If  we 


INTERACTION  BETWEEN  ANTIGEN  AND  ANTIBODY     153 

regard  the  action  of  the  amboceptor-complement  combination  upon 
the  albuminous  antigen  as  comparable  to  the  digestion  of  proteins 
by  the  digestive  ferments  of  the  gastro-intestinal  tract,  in  other 
words,  as  a  parenteral  digestion,  then  we  can  also  suppose  that  the 
anaphylactic  poison  represents  some  cleavage  product  of  the  protein 
molecule.  As  a  matter  of  fact,  there  is  a  certain  similarity  of  the 
symptoms  of  the  anaphylactic  shock  in  dogs  to  what  we  see  in 
"peptone"  poisoning  in  the  same  animal.  In  both  instances  there 
is  a  marked  drop  in  blood  pressure,  incoagulability  of  the  blood  and 
leukopenia,  and  in  both  cases  it  is  possible  to  counteract  the  poison- 
ous effect  by  the  administration  of  barium  chloride.  In  other 
animals,  however,  such  as  the  guinea-pig,  "peptone"  apparently 
plays  little  or  no  role;  Witte  peptone,  indeed,  is  quite  harmless  for  this 
animal,  which,  after  all,  is  the  most  sensitive  to  anaphylactic  shock. 
Barium  chloride,  moreover,  does  not  prevent  the  latter,  and  a  primary 
drop  in  blood  pressure,  such  as  we  see  in  dogs,  does  not  occur.  But 
it  is  conceivable  that  while  "peptone"  does  not  play  an  important 
role,  if  indeed  any,  that  other  poisonous  substances  may  be  formed 
which  may  be  quite  harmless  for  the  dog,  but  highly  toxic  for  the 
guinea-pig. 

Whether  or  not  the  anaphylactic  poisons  which  are  split  off  from 
different  antigens  by  the  antiserum  of  a  given  animal  or  animal 
species  are  identical  is  unknown,  but  does  not  seem  unlikely  in 
view  of  the  uniformity  of  the  anaphylactic  symptom  complex. 

In  speaking  of  the  anaphylactic  poison  in  the  foregoing  pages 
we  have  repeatedly  made  use  of  the  term  anaphylatoxin,  which 
has  come  into  common  use  so  extensively  that  it  would  indeed  be 
difficult  to  replace  it.  This  term,  of  course,  suggests  that  the  poison 
actually  belongs  to  the  class  of  toxins  which,  as  we  have  seen,  are 
characterized  by  the  fact  that  on  immunization  they  give  rise  to 
a  corresponding  antitoxin.  As  yet  there  is  no  evidence,  however, 
to  show  that  it  is  possible  to  immunize  against  this  poison,  and  it 
would  accordingly  be  better  not  to  use  the  term  anaphylatoxin  at 
all,  or  if  so,  to  bear  in  mind  that  by  "toxin"  in  this  case  we  merely 
mean  a  poison  in  the  more  general  sense  of  the  word. 

Seat  of  Interaction  between  Antigen  and  Antibody. — While  in  the 
past  it  seems  to  have  been  assumed  by  the  majority  of  observers 
that  the  interaction  between  antigen  and  its  anaphylactic  antibody 
takes  place  in  the  circulation,  evidence  has  of  late  been  adduced 


154  ANAPHYLAXIS 

which  suggests  that  the  anaphylactic  reaction  may  be  brought 
about  in  consequence  of  an  interaction  between  sessile  antibodies, 
i.  e.,  antibodies  which  are  still  in  union  with  the  cells  which  gave  rise 
to  their  formation,  and  the  corresponding  antigen.  This  view  is 
supported  by  the  experiments  of  Schultz  and  notably  of  Dale  on  the 
one  hand,  and  of  Manwaring,  Voegtlin,  and  Bernheim  on  the  other. 
Dale  thus  showed  that  the  virgin  uterus  of  sensitized  guinea-pigs, 
after  being  freed  from  serum  by  thorough  perfusion  with  Locke's 
solution,  will  exhibit  a  definite  rise  of  tonus  in  response  to  extreme 
dilutions  of  the  respective  antigen,  and  that  one  dose  of  the  specific 
antigen,  in  sufficient  concentration  to  produce  a  "maximal"  response 
of  the  anaphylactic  plain  muscle,  completely  desensitizes  the  latter 
to  further  doses  of  any  dimensions.  Corresponding  results  were 
obtained  with  the  isolated  lungs  of  the  animal  after  perfusion  with 
Ringer's  solution.  While  in  the  guinea-pig  the  localization  of  the 
anaphylactic  reaction  in  the  plain  muscle  tissue  has  thus  been 
established,  Manwaring,  Voegtlin,  and  Bernheim  have  conclusively 
demonstrated  that  in  the  dog  the  primary  effect  takes  place  in  the 
liver.  If  the  liver  of  a  sensitized  dog  is  thus  excluded  from  the 
general  circulation,  the  reinjection  of  the  corresponding  antigen 
does  not  produce  shock;  if,  however,  at  this  time  the  clamp  on  the 
hepatic  artery  is  removed  anaphylactic  symptoms  promptly  develop. 

While  these  experiments  of  course  strongly  suggest  the  existence 
of  sessile  anaphylactic  antibodies  in  the  tissues  which  have  been 
studied,  it  does  not  follow  that  an  interaction  between  antigen  and 
antibody  may  not  also  occur  in  the  circulation  with  consequent  pro- 
duction of  shock.  That  the  anaphylactic  antibody  finds  its  way  into 
the  circulation  can  hardly  be  disputed,  and  one  could  conceive  that 
under  certain  quantitative  conditions  it  could  here  play  the  role  of 
an  antitoxin.  This  possibility  has  indeed  not  yet  been  satisfactorily 
worked  out.  At  the  same  time  one  could  also  imagine  that  this 
combination  is  at  first  only  a  loose  one  and  that  it  might  yet  be  broken 
by  the  sessile  antibodies  with  consequent  anaphylactic  shock. 
Opposed  to  the  idea  that  the  free  antibody  might  play  the  role  of  an 
antitoxin,  i.  e.,  of  a  protective  substance,  is  the  fact  that  as  a  conse- 
quence of  the  interaction  between  antigen  and  antibody  in  the  test- 
tube,  a  reaction  product  develops  which  on  injection  into  the  normal, 
non-sensitized  animal  will  develop  anaphylactic  symptoms. 

Further  work  along  these  lines  will  be  necessary  before  a  true 


MECHANISM  OF  THE  ANAPHYLACTIC  SHOCK  155 

picture  of  the  proceedings  can  be  developed,  but  the  work  of  Schultz, 
Dale,  Manwaring,  Voegtlin,  and  Bernheim  mark  a  most  important 
advance  and  clearly  show  the  direction  in  which  profitable  research 
may  be  pursued. 

Mechanism  of  the  Anaphylactic  Shock. — If  now  we  inquire  into 
the  mechanism  by  which  the  anaphylactic  shock  is  called  forth,  the 
very  suddenness  of  the  onset  and  the  lightning  course  of  the  reaction 
suggest  a  cerebral  origin  of  the  symptoms  in  question.  As  a  matter 
of  fact  Besredka  has  shown  that  the  shock,  in  guinea-pigs  at  least, 
is  particularly  severe  if  the  anaphylactic  poison  is  injected  intra- 
cerebrally,  and  that  it  is  then  exceptional  for  an  animal  to  escape 
death,  while  with  the  usual  intraperitoneal  method  nearly  75  per 
cent,  recover.  Quite  in  accord  with  this  view  also  is  the  observation 
that  it  is  possible  either  to  suppress  or  to  mitigate  the  severity  of 
the  symptoms,  if  the  animal  is  previously  anesthetized  with  ether 
or  ethyl  chloride,  or  is  treated  with  hypnotics,  such  as  urethane, 
paraldehyde,  or  chloral  hydrate. 

Biedl  and  Kraus,  on  the  other  hand,  working  with  dogs,  came  to 
the  conclusion  that  primary  injury  to  the  brain  can  probably  be 
excluded,  since  paralysis  and  respiratory  disturbances  were  not 
observed  and  the  reflexes  did  not  disappear.  They  could  note  as 
a  constant  symptom,  however,  a  marked  drop  in  blood-pressure 
(from  120  to  150  Hgmn.  to  80,  60,  and  even  40),  which  was  shown 
to  be  of  peripheral  origin,  and  they  are  inclined  to  attribute  prac- 
tically all  other  symptoms,  which  have  been  noted  during  the 
anaphylactic  reaction,  including  the  drop  in  temperature  which  is 
seen  in  all  cases,  to  this  one  factor.  The  effect  of  the  narcotics  and 
hypnotics  they  explain  by  the  assumption  that  these  remedies 
merely  render  the  central  nervous  system  less  susceptible  to  the 
effect  of  stimuli  resulting  from  the  drop  of  blood-pressure  and  the 
consequent  central  anemia. 

Auer  and  Lewis  also  assume  a  peripheral  origin  of  the  anaphyl- 
actic symptom  complex,  but  regard  the  spasmodic  contraction  of 
the  smallest  bronchioles  which  is  so  constantly  seen  in  guinea-pigs 
as  the  essential  factor,  in  these  animals  at  least.  It  would  thus 
appear  that  in  different  animals  the  mechanism  may  be  different, 
but  the  possibility  must  also  be  borne  in  mind  that  these  differences 
may  be  more  or  less  accidental  and  not  essential.  This  idea  is 
supported  by  the  observation  of  Schultz  and  Jordan  that  the  bron- 


156  ANAPHYLAXIS 

chial  mucous  membrane  of  guinea-pigs  is  especially  thick  and  folded 
in  such  a  manner  that  relatively  slight  contractions  of  the  muscle 
fibers,  which  in  other  animals  would  lead  to  no  untoward  results, 
might  be  sufficient  in  the  guinea-pig  to  effect  complete  occlusion 
of  the  bronchial  lumen.  Evidently,  however,  our  knowledge  of 
existing  conditions  is  as  yet  too  meager  to  warrant  any  far-reaching 
conclusions. 

Mechanism  of  Antianaphylaxis. — As  regards  the  mechanism  which 
underlies  the  production  of  antianaphylaxis.  Friedberger  has  sug- 
gested that  it  might  be  due  to  the  absorption  or  neutralization 
(sc.,  inactivation)  by  the  second  dose  of  antigen  of  any  antibody 
that  may  be  present  at  the  time,  so  that  a  subsequent  injection  of 
antigen  does  not  meet  with  enough  antibody  to  form  a  fatal  dose 
of  poison.  Antianaphylaxis  will  hence  be  observed  during  the  pre- 
susceptible  stage,  because  not  enough  antibody  has  as  yet  been 
formed  and  during  the  susceptible  stage  (if  a  subfatal  dose  of  anti- 
gen is  injected),  because  all  or  most  of  the  antibody  is  used  up  by 
the  subfatal  dose  of  the  antigen.  This  principle  is  now  practically 
utilized  when  the  necessity  arises  of  reinjecting  a  patient  who  has 
been  sensitized  by  a  previous  injection  of  alien  serum  (antitoxin) .  The 
individual  then  receives  a  very  small  dose  of  the  serum  to  be  admin- 
istered about  an  hour  or  two  before  the  full  dose  is  given, 
or  the  serum  may  be  administered  so  slowly  that  the  antianaphyl- 
actic  state  can  actually  develop  during  the  injection  (see  Adminis- 
tration of  Diphtheria  Antitoxin). 

Friedberger's  explanation  of  the  mechanism  underlying  the  devel- 
opment of  antianaphylaxis,  as  outlined  above,  is  in  perfect  accord 
with  the  experimental  facts,  such  as  its  abrupt  development,  its 
specificity  and  even  with  the  apparently  contradictory  observation 
that  anaphylactic  susceptibility  can  be  passively  transferred  to 
another  animal  even  during  the  antianaphylactic  state  (as  a  portion 
of  the  anaphylactic  antibody  may  have  escaped  neutralization). 
As  Pfeiffer  and  Mita,  moreover,  have  shown,  the  proteolytic  fer- 
ment which  may  be  demonstrated  in  the  serum  of  the  sensitized 
animal,  disappears  with  the  development  of  antianaphylaxis. 

Strongly  in  support  of  Friedberger's  view  also  is  the  fact  that 
sensitized  animals  which  have  been  rendered  antianaphylactic 
with  the  homologous  serum  are  thereby  not  protected  against  the 
anaphylatoxin  itself. 


MECHANISM  OF  ANTIANAPHYLAXIS  157 

Not  to  be  confounded  with  the  antianaphylactic  state  proper 
is  the  protection  which  may  be  afforded  the  sensitized  animal  by 
various  other  procedures,  such  as  the  injection  of  peptone,  of  heterol- 
ogous  alien  sera,  and  even  of  indifferent  inorganic  substances  like 
kaolin.  Such  protection  in  contradistinction  to  true  antianaphyl- 
axis  is,  however,  not  specific,  does  not  develop  abruptly,  and  rapidly 
disappears  while  true  antianaphylaxis  persists  for  a  much  longer 
time,  and  is  within  quantitative  limits  absolutely  specific. 


CHAPTER  XI 
ANAPHYLAXIS  IN   ITS  RELATION  TO  DISEASE 

THE  discovery  that  the  parenteral  introduction  of  foreign  albumins 
into  the  animal  body  leads  to  an  anaphylactic  state,  in  consequence 
of  which  the  reintroduction  of  the  corresponding  substances  is  fol- 
lowed by  changes  which  are  of  more  or  less  serious  effect  upon  the 
body  at  large,  has,  of  course,  raised  the  question,  whether  certain 
symptoms  which  we  observe  in  the  course  of  the  various  infectious 
diseases  may  not  be  anaphylactic  in  origin  and  whether  certain  non- 
infectious  diseases  may  not  be  referable  to  such  factors  altogether. 

A  study  of  the  various  diseases  from  this  standpoint  has  elicited 
a  number  of  interesting  data,  though  we  must  admit  that  our 
knowledge  of  these  questions  has  not  extended  very  far  beyond 
the  domain  of  possibilities. 

The  earliest  investigations  in  this  direction  we  owe  to  v.  Pirquet 
and  Schick,  and  from  these  we  are  unquestionably  justified  in 
inferring  that  the  second  possibility  above  mentioned  actually  exists, 
viz.,  that  diseases  occur  which  may  be  wholly  due  to  the  existence 
of  an  anaphylactic  state  in  reference  to  certain  proteins.  The 
most  notable  example  of  this  order  is  the  serum  sickness  which  is 
observed  in  certain  individuals,  following  the  injection  of  the  various 
antitoxic  and  bacteriolytic  sera,  and  which,  as  we  have  already 
pointed  out,  is  not  referable  to  the  contained  antibodies,  but  to 
the  albumins  of  the  alien  sera  in  themselves.  The  picture  which 
here  develops  is  in  many  respects  very  similar  to  what  we  see  in 
certain  infectious  diseases,  although  the  material  which  is  intro- 
duced is,  of  course,  sterile.  Here,  as  there,  the  clinical  symptoms 
do  not  appear  at  once,  but  only  after  a  certain  interval,  which  is 
quite  analogous  to  the  so-called  period  of  incubation  of  the  infectious 
diseases. 

This  observation  is  very  important,  as  it  has  thrown  a  new  light 
upon  the  occurrences  in  the  body  during  that  period  and  upon  the 
manner  in  which  some  of  the  clinical  symptoms  of  the  infectious 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE  159 

diseases  may  originate.  In  the  past  we  have  looked  upon  the 
"period  of  incubation"  as  representing  a  period  of  time  during 
which  the  infecting  organisms  multiply  in  the  body  of  the  infected 
individual  to  that  point  at  which  they  would  be  sufficiently  numerous 
to  give  rise  to  symptoms  of  disease,  either  through  their  toxins 
(sc.,  endotoxins)  or  through  interference  with  the  metabolism  of 
the  macro  organism  in  other  ways.  This  explanation,  however, 
is  manifestly  out  of  the  question  in  accounting  for  the  "period  of 
incubation"  which  precedes  the  development  of  the  serum  sickness 
where  no  infecting  organisms  are  at  work.  But  v.  Pirquet  has  pointed 
out  that  the  phenomenon  is  readily  accounted  for,  if  we  bear  in 
mind  that  during  this  period  antibody  formation  is  taking  place, 
and  that  an  antibody-antigen  reaction  will  occur,  as  soon  as  the 
former  has  progressed  to  a  certain  point. 

This  point  we  may  well  term  the  threshold  of  anaphylactic  or  more 
generally  speaking  of  allergic  reaction.  If  at  this  time  the  antigen 
— in  the  present  instance  the  albumins  of  the  horse  serum — has 
disappeared  from  the  circulation,  no  symptoms  will,  of  course, 
result;  if,  however,  some  of  the  material  is  still  present,  a  reaction 
occurs,  during  which,  as  we  now  know,  poisonous  substances  (ana- 
phylatoxins,  apotoxins)  are  produced,  and  to  these  in  turn  we  may 
logically  attribute  the  symptoms  which  then  develop.  The  occur- 
rences just  described  may  be  diagrammatically  represented,  as 
shown  in  Fig.  2. 

If,  following  the  first  injection  of  horse  serum,  a  certain  interval 
be  allowed  to  elapse,  and  a  second  injection  be  then  given,  the 
result  will  differ  from  the  first  not  only  in  point  of  time  of  reaction, 
but  also  qualitatively  and  quantitatively,  so  far  as  the  symptoms 
are  concerned.  If  the  second  injection  be  given  at  a  time  when 
antibodies  are  still  present  in  the  circulation  in  considerable  amount, 
a  reaction  will  occur  either  immediately  or  within  the  first  twenty- 
four  hours;  this  may  be  quite  violent  in  its  intensity,  though  its 
duration  is  shorter  than  in  the  first  instance.  This  immediate  reaction 
is  also  diagrammatically  represented  in  Fig.  2. 

If,  on  the  other  hand,  the  second  injection  be  given  after  several 
years,  i.  e.,  at  a  time  when  the  antibodies  called  forth  by  the  first 
injection  have  disappeared,  a  certain  interval  of  time  will  elapse 
before  symptoms  of  serum  sickness  develop,  as  in  the  case  of  the 
first  injection.  But  whereas  this  interval  in  the  first  instance  is 


160 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 


I/ 


usually  from  eight  to  twelve  days  we  now  find  that  symptoms 
appear  after  from  four  to  seven  days.     Reactions  of  this  order 


Days  I 


Horse 

Serum 

(Allergen) 


FIG.  2 
15 


First  Injection 


.22 


29 


Period  of  Serum 

Incubation          Sickness 


Second  Injection  of 
Horse  Serum 


rgin 


Immediate 
Reaction 


Diagram  representing  the  interaction  between  horse  serum  and  the  corresponding  ergin  in 
relation  to  the  development  of  serum  sickness  and  the  occurrence  of  an  immediate  reaction. 
(Taken  from  v.  Pirquet.) 


FIG.  3 


Allergen 

(Second 

injection) 


Days  1  8 


Brief      Seru 
incubation  Sickness 
period 

Diagram  representing  the  interaction  be- 
tween horse  serum  and  the  corresponding  ergin 
in  relation  to  the  occurrence  of  a  double  reac- 
tion, i.  e.,  an  immediate  followed  by  a  hastened 
reaction.  (Taken  from  v.  Pirquet.) 


Immediate     Hastened 
Reaction       Reaction 

Diagram  representing  the  interaction  be- 
tween horse  serum  and  the  corresponding 
ergin  in  relation  to  the  occurrence  of  a  hastened 
reaction.  Note  the  greater  depth  of  the 
antibody  fraction  as  compared  with  the 
preceding  figure.  (Taken  from  v.  Pirquet.) 


v.  Pirquet  speaks  of  as  hastened  reactions.  They  are  readily  accounted 
for  if  we  remember  that  a  cell  which  has  once  been  stimulated  to 
active  antibody  formation  (sc.,  liberation)  will  subsequently  respond 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE  161 


to  the  same  stimulus  with  increased  activity.     This  may  be  dia- 
grammatically  represented,  as  shown  in  Fig.  3. 

Theoretically  we  should  expect  another  possibility  to  exist,  viz., 
the  occurrence  of  an  immediate,  followed  by  a  hastened  reaction,  as 
the  result  of  a  second  injection.  This  may  actually  occur,  and  is 
readily  explained  by  the  assumption  that  at  the  time  of  the  second 
injection  a  small  amount  of  antibody  was  still  present,  but  that 
this  was  not  sufficient  to  satisfy  the  affinities  of  the  total  amount 
of  albumin  introduced;  that  a  portion  of  the  latter  hence  calls  forth 
the  production  of  an  additional  amount  of  antibody  which  occurs 
in  a  "  hastened' '  manner,  and  meeting  with  some  of  the  free  antigen 
gives  rise  to  the  hastened  reaction,  as  shown  in  Fig.  4. 

If  now  we  compare  these  findings  with  certain  occurrences  which 
may  be  observed  in  connection  with  some  of  the  infectious  diseases, 
it  will  be  seen  that  the  appearance  of  certain  symptoms  which  we 
note  in  some  of  the  latter,  may  readily  be  explained  upon  the  same 
basis  as  the  reactions  which  follow  reinjections  of  horse  serum,  as 
above  outlined,  so  that  the  inference  seems  justifiable  that  the 
underlying  mechanism  in  the  two  groups  of  cases  is  also  essentially 
the  same. 

As  is  well  known  a  first  vaccination  with  cowpox  lymph  is  followed 
by  a  period  of  seven  or  eight  days,  during  which  there  is  a  slowly 
developing  local  reaction  without  any  noticeable  systemic  symptoms. 
During  the  first  two  days  the  local  response  is  evidently  purely 
traumatic  in  character.  On  the  third  or  fourth  day  the  specific  reac- 
tion begins  in  the  form  of  a  small  papular  elevation  which,  between 
the  fourth  and  sixth  days,  is  then  differentiated  into  a  central 
papilla  and  a  surrounding  areola.  Up  to  the  eighth  day  the  latter 
extends  but  slightly  beyond  the  papilla,  but  between  this  and  the 
eleventh  day  it  rapidly  develops  so  as  to  form  a  well-marked  inflam- 
matory zone  surrounding  the  central  area,  reaching  its  largest  size 
between  the  eleventh  and  the  fifteenth  day.  After  this  it  gradually 
disappears,  while  the  papilla  dries  up  and  exfoliates.  Coincidently 
with  the  development  of  the  areola,  there  are  frequently  also  systemic 
symptoms,  of  which  fever  and  leukopenia  are  the  most  striking. 

If  now  we  compare  this  picture  with  that  of  serum  sickness,  we 

find  very  striking  points  of  similarity  which  strongly  suggest  that 

the  underlying  mechanism  is  in  all  probability  the  same.    Here,  as 

there,  we  have  a  period  of  incubation  of  virtually  the  same  duration. 

11 


•i! 


162 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 


But,  whereas  the  injection  of  horse  serum  does  not  necessarily  give 
rise  to  any  symptoms  during  this  time,  since  the  material  that  is 
introduced  is  sterile,  vaccination  is  early  followed  by  certain  local 
symptoms  which  we  may  logically  attribute  to  a  multiplication  of 
the  organism  of  cowpox  in  the  skin.  This  is  shown  diagrammatically 
in  Fig.  5  in  the  gradually  ascending  line  representing  the  first  vac- 
cination. We  may  then  suppose  that  the  absorption  of  some  dead 
organisms  (dead  when  introduced  or  destroyed  by  the  local  defen- 
sive forces  shortly  after  their  introduction),  i.  e.,  of  their  proteins, 


FIG.  5 


Days}l  ......   ,8 


15 


Injection  of 
larger  quantity 
of  Vaccine 


Incubation 
period 


Intense  local  and 
general  reaction 


Diagram  illustrating  the  effect  of  vaccination  in  its  relation  to  antibody  formation  upon  the 
development  of  the  corresponding  clinical  symptoms.     (Taken  from  v.  Pirquet.) 

is  followed  after  the  usual  period  of  about  eight  days  by  the  pro- 
duction of  the  corresponding  antibodies,  some  of  which,  no  doubt, 
bring  about  the  destruction  of  all  the  remaining  organisms,  while 
others  react  with  the  liberated  proteins  and  give  rise  to  anaphylatoxins 
which  in  turn  are  responsible  for  the  rapidly  developing  local  inflam- 
matory reaction,  as  also  perhaps  for  some  of  the  systemic  symptoms. 
Theoretically,  of  course,  the  anaphylactic  response  should  continue 
so  long  as  both  antigen  and  antibody  are  present,  a  conclusion  with 
which  clinical  observation  is  in  perfect  accord.  It  might,  of  course, 
be  argued  that  the  period  of  incubation  following  vaccination  was 
after  all  due  to  the  multiplication  of  the  variola  organisms,  and  that 
so  soon  as  this  had  exceeded  a  certain  point,  the  local  as  well  as  the 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE          163 

general  symptoms  would  have  occurred  irrespective  of  any  antibody 
production.  This  conclusion,  however,  is  disproved  by  the  fact 
that  the  size  of  the  vaccine  dose  neither  hastens  nor  retards  the  devel- 
opment of  the  symptoms,  and  is  further  especially  strikingly  demon- 
strated in  experiments  of  v.  Pirquet,  where  the  same  patient  was 
vaccinated  on  successive  days.  When  this  was  done  all  points  of 
vaccination  developed  their  areola  on  the  same  day,  so  that  in  a 
given  instance  when  the  first  vaccination  reached  its  inflammatory 
maximum  after  eleven  days  the  subsequent  vaccinations  were  equally 
advanced  after  ten,  eight,  and  four  days. 

In  this  connection  it  is  interesting  to  note  that  whereas  the  vacci- 
nated individual  has  acquired  a  marked  immunity  to  infection  with 
the  organism  of  either  human  smallpox  or  cowpox,  he  is,  never- 
theless, hypersensitive  to  its  proteins,  v.  Pirquet  thus  remarks 
that  by  frequently  vaccinating  himself  he  brought  the  skin  of  his 
forearm  to  such  a  state  of  hypersensitiveness  that  after  twelve  hours 
a  papule,  measuring  on  an  average  9  mm.  in  diameter,  i.  e.,  a  size 
only  reached  in  primary  vaccinations  on  the  seventh  day,  develops. 
This,  of  course,  is  closely  analogous  to  what  we  see  on  reinjection 
with  horse  serum  during  the  first  six  months  following  the  primary 
injection,  and  represents  what  v.  Pirquet  terms  a  hastened  reaction. 
An  immediate  reaction  is  here  not  observed,  probably  because  it  is 
obscured  by  the  traumatic  reaction. 

If  now  we  apply  the  same  principle  to  the  study  of  tuberculosis 
it  will  be  seen  that  here  also  various  phases  of  the  disease  can  be 
satisfactorily  explained  upon  the  same  basis  (v.  Pirquet) .  In  experi- 
mental tuberculosis,  produced  in  cattle,  a  quiescent  period  of  "incu- 
bation," extending  over  eight  days,  likewise  follows  the  inoculation, 
provided  that  the  initial  infecting  dose  was  sufficiently  large,  exactly 
as  in  connection  with  vaccination  and  the  development  of  primary 
serum  sickness  (Fig.  6).  Coincidently  with  the  appearance  of 
antibodies  clinical  symptoms  then  develop;  but  whereas  in  vaccina- 
tion the  production  of  antibodies  leads  to  the  prompt  destruction 
of  the  invading  organism,  the  tubercle  bacilli,  owing  to  their  peculiar 
waxy  envelope,  no  doubt  succeed  in  maintaining  themselves  in  the 
body  of  the  infected  organism,  and  may,  if  their  initial  number  was 
sufficiently  large,  even  cause  the  death  of  the  host.  A  certain  num- 
ber, of  course,  are  destroyed,  and,  as  protein  antigen  and  antibody 
thus  coexist,  a  more  or  less  continuous  formation  of  anaphylatoxin 


164 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 


takes  place,  and  becomes,  in  turn,  to  a  certain  extent  at  least,  respon- 
sible for  the  more  or  less  continuous  symptomatic  evidence  of  disease. 
If  the  number  of  organisms  is  small,  or  if  they  have  been  attenu- 
ated by  artificial  means,  the  incubation  period  is  much  longer.  In 
such  an  event  the  antibody  production  begins  in  the  third  week, 
but  it  is  not  until  the  fifth  week  that  a  sufficient  quantity  of 
anaphylatoxin  is  formed  to  elicit  manifest  symptoms  (Fig.  7). 


Fia.  6 


15 


Threshold  of  Death- 


Injection  of  virulent 
|     tuberclebacilli 


Manifestations  of  Disease 

Diagram  illustrating  the  interaction  between  antigen  and  antibody  in  a  fatal  case  of  cattle 
tuberculosis,  following  the  injection  of  a  moderate  dose  of  tubercle  bacilli.    (Taken  from  v.  Pirquet.) 


Days  I  f 

FIG.  7 
8                         15 

-1  —  1  —  1  —  1  —  I  —  !  —  1  —  I      i      i      1      i      I      i      i 

22 
.    i    i     .    .    . 

29 

Threshold  of  clinical  evidence  of  d 


..  Clinical 

Symptoms 

Diagram  illustrating  a  protracted  period  of  incubation  in  its  relation  to  the  interaction  between 
tubercular  antigen  and  the  corresponding  antibody;  infection  having  been  produced  by  the  admin- 
istration of  tubercle  bacilli  in  small  number  or  in  attenuated  condition.  (Taken  from  v.  Pirquet.) 

The  subsequent  course  of  the  infection  will,  of  course,  depend 
upon  circumstances.  If  recovery  takes  place  the  further  multipli- 
cation of  tubercle  bacilli  ceases;  the  foci  that  are  already  in  exist- 
ence are  encapsulated  and  the  active  clinical  symptoms  disappear. 


ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 


165 


But,  as  is  shown  in  Fig.  8,  antibodies  still  remain  in  considerable 
amount,  so  that  any  factor  which  would  now  call  a  latent  tubercular 
focus  into  renewed  activity,  or  the  introduction  of  tuberculin  from 
without,  would  also  call  forth  a  prompt  clinical  reaction,  either 


Mon.l          2         3,4         5 , 


Fio.  8 
7     .     8 


.    33 


153  Months 


Threshold  of 
Sub&itaneov* 

'reaction 


Threshold  of 

Cutaneous 

reaction 


Symptoms  of  Disease 
Diagram  illustrating  benign  course  in  a  case  of  human  tuberculosis.    (Taken  from  v.  Pirquet.) 


Threshold  of  Clinical  evidence 


FIG.  9 

Days  after  appearance  of  eruption  in  measles 
.    .   .    .   I I1 I8.    .    .  .. 


Death- 


Measles 


Miliarv  Tuberculosis 


22    24 


Diagram  illustrating  the  lighting  up  of  a  tubercular  process  (miliary  tuberculosis)  following 
measles.     (Taken  from  v.  Pirquet.) 

general  or  local,  as  the  case  may  be.  Fig.  9  illustrates  this  very  well, 
and  shows  the  mechanism  which  is  called  into  action,  when  a  miliary 
tuberculosis  follows  an  attack  of  measles,  in  a  subject  having  a  latent 
(inactive)  tubercular  infection. 


166  ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 

If  now  we  pass  on  to  a  consideration  of  an  infection  with  an 
organism  which  is  a  pure  toxin  producer  the  interpretation  of  the 
clinical  picture  will  be  different.  In  diphtheria,  for  example,  we  have 
coincidently  with  the  multiplication  of  the  invading  organisms 
a  production  of  toxin.  This  in  itself  is,  of  course,  quite  sufficient  to 
account  for  practically  all  the  clinical  symptoms  that  we  observe. 
These  set  in  early,  since  comparatively  few  organisms  are  capable 
of  producing  toxin  in  sufficient  amount  to  call  forth  clinical  evidence 
of  disease.  There  is  hence  not  the  usual  incubation  period  of  eight 
days,  and  the  initial  symptoms  in  any  event  are  not  due  to  any 
antigen-antibody  reaction,  but  to  the  toxin  itself.  When  the  anti- 
bodies then  appear  we  may,  of  course,  rightfully  assume  that  pre- 
cipitins  are  formed,  as  well  as  lysins  and  antitoxins,  and  theoretically 
we  might  expect  a  clinical  reaction  due  to  anaphylatoxins.  Clini- 
cally, however,  we  have  no  clear  evidence  of  this,  which  is  probably 
owing  to  the  fact  that  the  toxin  effect  by  itself  controls  the  entire 
picture.  In  scarlatina,  v.  Pirquet  concedes  that  the  primary  malady, 
i.  e.,  the  eruptive  fever  per  se,  is  similarly  a  pure  toxin  effect,  but 
that  the  sequelae,  and  notably  the  nephritis,  are  the  expression  of  the 
action  of  anaphylatoxins,  which  are  formed,  if  at  a  time  when  the 
corresponding  antibodies  are  present,  an  autoreinfection  (from 
a  broken-down  lymph  gland  for  example)  occurs.  A  toxin  effect, 
of  the  primary  type,  is  then  not  produced,  since  antitoxins  are  at 
the  time  present  in  sufficient  quantity  to  counteract  their  effect 
(Fig.  10). 

It  would,  of  course,  lead  too  far  to  continue  the  analysis  of  the 
different  infectious  diseases  along  these  lines,  but  I  believe  to  have 
shown  that  the  anaphylactic  principle  serves  to  explain  many  points 
in  clinical  symptomatology  for  which  an  adequate  explanation  has 
heretofore  been  lacking.  If  we  consider  that  the  absorption  of  alien 
proteins  (and  hence  of  bacterial  proteins)  probably  always  gives 
rise  to  the  production  of  corresponding  antibodies  of  the  anaphyl- 
actin  type,  we  can  also  understand  that  there  is  probably  not  a 
single  infectious  disease  in  which  they  are  not  formed,  and  in  which 
they  cannot,  theoretically  at  least,  play  an  active  part.  Besides 
the  diseases  already  discussed  this  would  certainly  seem  most  likely 
in  syphilis,  in  typhoid  fever,  measles,  glanders,  and  pneumonia,  and 
future  studies  of  these  diseases  from  this  standpoint  will,  no  doubt 
lead  to  interesting  results.  All  along  the  line  a  start  has  indeed  only 


IDIOSYNCRASIES  AND  ANAPHYLAXIS 


167 


now  been  made,  and  a  great  deal  remains  to  be  done,  but  I  believe 
that  there  is  scarcely  any  field  of  study,  which  from  the  standpoint 
of  the  clinician  promises  such  fruitful  returns,  as  the  investigation 
of  our  common  infectious  diseases  and  their  pathogenic  agents  along 
these  lines. 

At  the  present  state  of  our  knowledge  it  is,  of  course,  very  diffi- 
cult to  decide  which  symptoms  in  a  given  disease  are  due  to  bacterial 
toxins,  which  to  endotoxins,  which  to  ptomains,  and  which  to  ana- 
phylatoxins;  but  it  is  important  to  recognize  that  with  the  discovery 
of  the  latter  a  new  vista  has  been  opened  up,  along  which  we  can  see 
the  possible  manner  in  which  some  of  those  organisms  may  produce 


FIG.  10 


Days 


Autoreinfection 


Threshold  of 
Clinical 
anifestations 


Scarlatina 


Sequela 


Diagram  illustrating  the  interaction  between  antigens  and  antibodies  in  their  relation  to  the 
clinical  picture  and  a  sequela  of  scarlatina.       (Taken  from  v.  Pirquet.) 

symptoms  of  disease  and  even  death,  which  are  recognizedly  not 
toxin  producers,  and  whose  endotoxins  also  are  not  sufficiently  active 
to  cause  the  clinically  recognizable  results  of  infection.  This  is 
indeed  a  most  attractive  field  for  speculation,  but  as  a  further 
discussion  of  the  many  possibilities  and  problems  which  suggest 
themselves  in  this  connection  would  of  necessity  be  purely  theoretical 
in  character,  it  will  be  better  to  leave  this  for  some  future  occasion, 
when  actual  experimental  data  may  be  at  our  disposal. 

Idiosyncrasies  and  Anaphylaxis. — I  have  pointed  out  above  that  the 
development  of  a  definite  symptom-complex  following  the  parenteral 
introduction  of  horse  serum  (i.  e.,  of  alien  albumin)  suggests  the 
possibility  that  some  of  the  non-infectious  diseases  with  which  we 


168  ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 

have  long  been  familiar  may  possibly  be  due  to  similar  causes. 
Recent  investigations  have  shown  that  such  is  actually  the  case,  and 
with  the  recognition  of  this  possibility  an  unexpected  ray  of  light  has 
reached  one  of  the  darkest  corners  of  our  clinical  rubbish  room 
where  have  reposed  for  centuries  the  time-honored  and  mystic 
"  idiosyncrasies.' ' 

Especially  interesting  in  this  connection  are  the  observations  which 
have  been  made  in  the  so-called  "hay  fever ,"  or  pollen  disease,  as  it 
would  be  more  appropriate  to  term  the  malady.  As  is  well  known, 
certain  individuals  are  annually  attacked  with  irritation  of  the 
mucosa  of  the  nose,  giving  rise  to  paroxysms  of  sneezing,  and  later 
with  a  similar  irritability  of  the  pharynx  and  the  trachea,  leading 
to  asthmatic  disturbances  of  greater  or  less  severity.  The  occurrence 
of  these  attacks  is  intimately  associated  with  the  time  of  the  year  at 
which  certain  plants  (belonging  to  the  order  of  the  Graminese,  also 
Ambrosia  and  Solidago)  come  into  blossom,  and  is  due,  as  Elliothson 
already  pointed  out  in  1831,  to  the  absorption  of  some  constituent 
of  the  pollen  of  the  respective  plants.  Weichardt  and  Wolff-Eisner 
then  pointed  out  (1905  and  1906)  the  close  similarity  between 
the  symptom-complex  in  question  and  the  serum  sickness  of 
v.  Pirquet  and  Schick,  and  suggested  that  it  also  could  readily  be 
explained  upon  the  basis  of  anaphylaxis.  As  a  matter  of  fact  it  is 
possible  in  a  susceptible  individual  to  call  forth  a  typical  attack  at 
any  time  either  by  the  introduction  of  a  suspension  of  the  corres- 
ponding pollen  into  the  conjunctiva,  or  by  its  subcutaneous  applica- 
tion, in  a  manner  quite  analogous  to  the  tuberculin  test.  Here  as  there 
the  amount  of  material  which  will  suffice  to  bring  about  a  reaction 
is  remarkably  small.  Wolff -Eisner  thus  reports  that  a  typical 
response  will  follow  the  application  of  but  two  drops  of  a  0.2  per 
cent,  solution  of  pollen,  and  Lubbert  mentions  that  in  highly  sus- 
ceptible individuals  TUiTTnT  milligram  even  may  produce  symptoms. 

Closely  related  to  pollen  fever  are  no  doubt  also  those  curious 
asthmatic  conditions  which  have  been  noted  in  some  individuals 
following  the  inhalation  of  Witte  peptone,  after  the  ingestion  of 
egg  albumen,  strawberries,  blueberries,  gooseberries,  various  legu- 
minous vegetables,  lobster,  chocolate,  cheese,  as  also  on  exposure  to 
certain  exhalations,  such  as  those  of  horses,  in  connection  with  attacks 
of  constipation,  etc.  To  the  same  category  evidently  belong  also 
those  curious  cases  of  diarrhea  which  in  some  persons  follow  the 


IDIOSYNCRASIES  AND  ANAPHYLAXIS  169 

ingestion  of  egg  albumen,  and  in  certain  babies  the  administration 
of  cows'  milk;  further,  also,  the  remarkable  symptom-complex, 
which  is  usually  designated  as  angioneurotic  edema,  the  long  list 
of  urticarias  which  follow  the  ingestion  of  lobster,  fish,  oysters, 
cheese,  and  strawberries;  certain  bacterial  exanthemata,  certain  skin 
affections  of  pregnancy,  the  dermatitis  of  satin-wood- workers,  the  phe- 
nomena of  fagopyrismus  (buckwheat  poisoning),  certain  anomalous 
drug  reactions,  etc. 

Here  we  have  entered  the  very  midst  of  the  idiosyncrasies  which 
in  former  years  seemed  shrouded  in  impenetrable  mystery,  and  which 
now,  in  view  of  our  knowledge  of  the  principles  underlying  anaphyl- 
axis,  seem  so  readily  accounted  for  on  this  basis,  and  as  merely 
being  the  expression  of  an  anomalous  reaction  on  the  part  of  certain 
individuals  to  the  parenteral  introduction  of  alien  proteins.  The 
question,  of  course,  still  remains  to  be  answered  why  one  person  and 
not  all  others  react  in  such  an  anomalous  manner  to  stimuli  which 
after  all  we  must  regard  as  normal.  At  the  present  we  can  merely 
theorize  on  these  points,  it  is  true,  but  we  can  do  so  with  the  knowl- 
edge that  we  have,  at  least,  a  basis  which  unquestionably  is  sound, 
and  the  time  is  evidently  not  far  off  when  this  chapter,  which  was 
only  a  few  years  ago  so  obscure,  will  be  one  of  the  best  understood 
in  physiological  pathology. 

This  is  not  the  place  to  enter  into  a  detailed  account  of  the  various 
idiosyncrasies  that  we  have  just  briefly  passed  in  review,  but  it  may 
be  permissible  nevertheless,  before  concluding  our  present  chapter, 
to  show  by  a  few  examples  that  we  have  already  gained  somewhat 
more  than  a  clinical  basis  for  our  belief  that  anaphylactic  action  is 
responsible  for  the  clinical  pictures  which  we  observe. 

Especially  interesting  in  this  connection  are  the  asthmatic  phe- 
nomena which  occur  in  some  persons  when  exposed  to  certain  exhala- 
tions. Remarkable  examples  of  this  order  have  been  described. 
Schittenhelm  thus  mentions  the  case  of  an  engineer  who  invariably 
was  attacked  with  "asthma"  when  he  was  obliged  to  enter  a  tunnel 
that  was  under  construction,  while  he  was  otherwise  free  from  any 
discomfort.  Another  person  was  subject  to  asthma  in  one  city  and 
not  in  another  only  a  few  miles  distant.  To  this  order  also  belong 
those  individuals  who  become  asthmatic  when  they  enter  a  horse- 
stable,  or  even  when  they  sit  in  a  carriage  behind  a  horse.  It  is 
interesting  to  note  that  a  number  of  persons  who  experienced  a 


170  ANAPHYLAXIS  IN  ITS  RELATION  TO  DISEASE 

severe  anaphylactic  attack  immediately  following  the  injection  of 
horse  serum  were  also  affected  by  the  exhalation  from  horses.  All 
such  cases  are  unquestionably  anaphylactic  in  character  and  refer- 
able to  the  absorption  of  organic  matter  that  is  present  in  the  ema- 
nations from  animals  and  human  beings.  This  explanation  seems 
reasonable  in  view  of  certain  findings  reported  by  Weichardt.  This 
observer  noted  that  guinea-pigs  which  had  been  injected  with  fluid 
through  which  the  expired  air  of  human  beings  had  been  passed, 
were  thus  rendered  anaphylactic  to  human  bronchial  secretion,  and 
on  intravenous  injection  with  the  latter  responded  with  a  marked 
drop  in  temperature  and  sopor. 

Especially  instructive  also  are  those  cases  in  which  the  ingestion 
of  such  common  articles  of  food  as  egg  albumen  and  cows'  milk  is 
followed  by  most  abnormal  consequences.  Landmann  thus  records 
a  case  where  the  ingestion  of  a  bit  of  egg  albumen,  no  larger  than  a 
pea,  was  followed  after  a  quarter  of  an  hour  by  a  burning  sensation 
and  swelling  of  the  tongue,  intense  edema  of  the  palate  and  pharynx, 
salivation,  lacrymation,  burning  and  itching  in  the  Eustachian 
tubes,  vomiting,  severe  diarrhea,  and  great  prostration,  while  its 
application  to  the  skin  resulted  in  urticaria.  Such  findings  are  quite 
analogous  to  the  anaphylactic  enteritis  which  may  be  observed  on 
injecting  sensitized  dogs  with  egg  albumen,  and  where  post  mortem 
the  mucosa  and  submucosa  of  the  entire  intestinal  tract  inclusive 
of  the  pylorus  is  studded  with  miliary  hemorrhages. 

In  a  case  of  so-called  fagopyrismus  (buckwheat  poisoning),  which 
unquestionably  also  belongs  to  this  order,  Thayer  had  the  patient 
vaccinated  with  the  substance  in  question,  the  result  within  half 
an  hour  being  a  feeling  of  oppression  and  nausea,  frequent  cough, 
a  rapid  and  intermittent  pulse,  suffusion  of  the  conjunctive,  ery- 
thema, intense  pruritus,  and  local  urticaria  at  the  point  of 
injection. 

In  a  case  of  antipork  idiosyncrasy,  Bruck  injected  some  of  the 
patient's  serum  into  a  guinea-pig  and  followed  this  up  with  an  injec- 
tion of  hog  serum,  the  result  being  a  typical  anaphylactic  shock, 
while  the  control  animals  showed  no  symptoms  whatever. 

Quite  recently  the  same  writer  has  further  shown  that  the  curious 
hypersusceptibility  which  certain  persons  show  toward  iodoform  can 
be  passively  transferred  in  the  individual's  serum  to  guinea-pigs, 
and  Klausner  could  demonstrate  that  this  was  possible  even  after 


IDIOSYNCRASIES  AND  ANAPHYLAXIS  171 

an  interval  of  fifteen  months  from  the  time  of  the  last  iodoform 
intoxication. 

I  myself,  while  suffering  from  a  trichinous  infection,  developed 
a  curious  type  of  dyspnea,  which  continued  for  a  number  of  months, 
and  which  I  am  now  strongly  tempted  to  look  upon  as  an  anaphyl- 
actic  reaction  due  to  the  absorption  of  trichinous  albumins. 

Evidently  this  is  a  most  interesting  chapter  in  our  modern  immu- 
nity work,  and  one  which  is  destined  to  assume  an  important  posi- 
tion in  clinical  medicine. 


CHAPTER  XII 
ACTIVE  IMMUNIZATION 

IF  now  we  pass  on  to  a  discussion  of  the  different  defensive  factors 
of  the  animal  body  from  the  standpoint  of  prophylactic  and  curative 
therapeutics  the  question  naturally  arises,  To  what  extent  have  we 
the  power  to  influence  this  mechanism  artificially?  In  view  of  the 
fact  that  we  have  scarcely  passed  farther  than  the  threshold  of  the 
study  of  immunology,  using  the  term  in  its  widest  sense,  it  is  natural 
that  our  attempts  to  utilize  principles  with  which  we  have  thus 
far  become  acquainted  should  have  been  relatively  crude,  and  that 
the  results  in  many  instances  have  not  led  to  a  satisfactory  end. 
An  enormous  amount  of  work  still  remains  to  be  done,  but  even  so  we 
have  every  reason  to  be  proud  of  what  has  already  been  achieved, 
and  to  believe  that  more  yet  will  be  accomplished  in  the  future. 

We  have  seen  that  even  under  normal  conditions  the  body  has  at 
its  disposal  defensive  forces  which  are  most  important,  and  which 
in  many  instances  are  quite  sufficient  to  prevent  a  general  infection, 
even  though  the  local  barriers  have  fallen.  The  battle  here  is,  no 
doubt,  frequently  won  before  specific  antibody  formation — our 
second  line  of  defence — has  even  begun.  For  many  centuries 
physicians  have  recognized  the  existence  of  so-called  predisposing 
causes  to  disease  and  their  influence  upon  the  course  of  the  individual 
case.  I  would  recall  the  effect  of  depressing  influences,  such  as  grief 
and  worry,  fatigue  and  hunger,  in  increasing  the  predisposition  to  a 
great  many  infectious  diseases. 

Quite  in  accord  with  clinical  observation  are  the  results  of  the 
animal  experiment.  Charrin  and  Roger  thus  succeeded  in  infecting 
rats  with  anthrax  after  they  had  been  greatly  fatigued  by  being 
made  to  run  in  a  tread-mill,  while  under  normal  conditions  the 
animals  are  quite  resistant.  Other  observers  could  break  the  natural 
immunity  of  dogs,  chickens,  and  pigeons  to  the  same  organism  by 
the  withdrawal  of  drinking  water;  in  pigeons  the  same  result  can  be 
obtained  by  fasting.  Quite  well  known  further,  both  clinically 


ACTIVE  IMMUNIZATION  173 

and  experimentally,  is  the  predisposing  influence  to  infection  of 
the  continued  use  of  alcohol  and  various  narcotics.  Of  the  manner 
in  which  these  agencies  bring  about  the  greater  susceptibility  to 
infection  nothing  was  known  in  the  past,  and  even  now  our  knowl- 
edge is  but  imperfect.  But  we  can  at  least  suggest  certain  possi- 
bilities. As  we  know  that  phagocytic  action  represents  one  of  the 
most  important  factors  in  our  first  line  of  defence,  and  as  this  has 
been  shown  to  depend  to  a  very  great  extent  upon  the  presence  of 
opsonins  and  tropins,  it  would  seem  reasonable  to  suppose  that  the 
harmful  agencies  just  referred  to  might  readily  operate  through 
interference  with  the  production  of  such  bodies  as  are  essential  to 
phagocytosis. 

In  this  connection  it  is  interesting  to  note  that  during  pregnancy 
which  has  long  been  recognized  as  a  factor  predisposing  to  the  devel- 
opment of  tuberculosis  the  opsonic  content  of  the  blood  tends  to 
be  abnormally  low  in  fully  50  per  cent,  of  the  cases.  Then,  again, 
we  can  conceive  that  the  normal  bacteriolytic  power  of  the  blood 
may  be  impaired  by  some  of  the  influences  in  question.  In  the  case 
of  chronic  alcoholism,  this  has  indeed  been  demonstrated  by  Abbot 
and  Bergey,  who  noted  that  there  was  a  diminution  of  complement. 

That  this  in  turn  may  actually  diminish  the  resistance  to  certain 
infections  has  been  shown  by  Pfeiffer  and  Moreschi.  These  investi- 
gators injected  a  series  of  guinea-pigs  intraperitoneally  with  a  fatal 
dose  of  cholera  vibrios  (equal  for  all  animals)  and  an  amount  of 
cholera  immune  serum  sufficient  to  protect  the  animals  against  the 
number  of  organisms  used.  At  the  same  time  they  received  varying 
amounts  of  normal  human  serum  and  a  constant  quantity  of  an  anti- 
human  rabbit  serum.  The  latter,  of  course,  contained  precipitins 
for  the  human  albumins,  and  the  idea  of  the  experiment  was  that 
as  a  consequence  of  the  interaction  between  precipitin  and  precipi- 
tinogen  (albumin),  and  the  resultant  formation  of  a  precipitate 
the  complement  of  the  guinea-pig  would  be  absorbed,  and  accord- 
ingly not  be  available  to  activate  the  anticholera  amboceptors,  so 
that  the  animal  would  lose  the  protective  influence  of  the  latter 
which  would  have  been  operative  had  complement  been  available. 
The  results  were  quite  in  accord  with  the  theoretical  demands, 
all  those  animals  having  died  in  which  occasion  for  complement 
elimination  was  afforded,  while  the  control  animals  which  had 
received  no  human  serum,  but  which  had  otherwise  been  treated 


174  ACTIVE  IMMUNIZATION 

in  the  same  manner,  lived.  Evidently  a  lack  of  complement  in  the 
course  of  an  infection  may  lead  to  disastrous  consequences,  and  the 
assumption  seems  justifiable  that  the  presence  of  an  insufficient 
quantity  at  the  start  may  favor  the  generalization  of  an  infection 
in  which  otherwise  a  local  reaction  only  might  have  taken  place. 

That  the  normal  amboceptors  might  be  similarly  influenced  is,  of 
course,  also  possible,  and  is  suggested  by  certain  experiments  of 
Abbot  and  Bergey,  who  found  that  the  hemolytic  amboceptors 
which  appear  in  the  guinea-pig  following  the  injection  of  alien  red 
corpuscles  rapidly  disappear  under  the  continued  administration 
of  alcohol. 

These  data,  few  as  they  are,  throw  some  light  upon  the  possible 
modus  operandi  of  some  of  the  causes  which  predispose  to  infectious 
diseases,  and  open  up  a  field  for  investigation  which,  speaking 
a  priori,  should  furnish  some  very  valuable  results.  Studies  in  this 
direction  would  also  show  by  what  general  non-specific  measures, 
dietetic,  medicinal,  or  otherwise,  the  resistance  against  infection 
could  be  raised,  and  the  likelihood  of  successful  specific  treatment 
thereby  enhanced.  At  the  present  time  the  latter  occupies  the  fore- 
ground of  medical  interest,  and  it  is  the  purpose  of  the  following 
pages  to  show  what  has  already  been  accomplished,  both  from  the 
standpoint  of  prophylaxis  and  of  treatment. 

In  arranging  the  sequence  of  our  subject  matter,  precedence  is 
given  to  those  methods  by  which  immunity  can  be  actively  produced, 
for  here  the  entire  defensive  mechanism  is  thrown  into  operation, 
whereas  in  the  production  of  passive  immunity  only  certain  indi- 
vidual defensive  principles  are  utilized. 


(A)    ACTIVE   IMMUNIZATION   FOR   PROPHYLACTIC   PURPOSES 

Since  the  entire  defensive  mechanism  of  the  animal  body  is  thrown 
into  action  as  a  result  of  active  immunization  it  would  suggest  itself 
that  attempts  in  this  direction  would  furnish  the  most  valuable 
results  when  employed  for  prophylactic  purposes.  When  infection 
has  once  taken  place,  and  clinical  symptoms  of  disease  have  already 
developed,  conditions  are  much  more  complicated.  The  effect  of 
toxins,  whether  produced  by  the  infecting  organisms  themselves,  or 
as  a  result  of  anaphylactic  reaction,  then  so  frequently  dominates 


SMALLPOX  175 

the  clinical  picture  that  sufficient  time  is  not  available  to  stimulate 
the  body  cells  to  active  immunization.  If  the  period  of  incubation 
of  a  given  malady  is  sufficiently  long,  so  as  to  permit  of  the  active 
mobilization  of  the  defensive  forces  before  symptoms  of  disease 
actually  develop,  attempts  at  active  immunization  would,  of  course, 
be  indicated,  and,  as  shown  in  our  prophylactic  treatment  of  rabies, 
may  give  rise  to  excellent  results.  Chronic  infections  further  would 
theoretically,  at  least,  lend  themselves  to  treatment  of  this  order, 
while  in  the  acute  maladies,  for  the  reasons  just  indicated,  we  can 
only  exceptionally  hope  to  exercise  a  favorable  influence  upon  the 
course  of  the  disease.  In  combination  with  the  use  of  antitoxic 
or  bacteriolytic  sera,  however,  it  might  even  then  be  tried. 

As  the  basis  of  all  attempts  at  active  immunization,  we  might 
very  appropriately  take  the  dictum:  that  there  can  be  no  protection 
without  infection,  bearing  in  mind,  however,  that  "infection"  is  not 
synonymous  with  "disease,"  that  "infection"  does  not  imply  a 
"virulent"  infection,  and  that,  immunologically  speaking,  the 
parenteral  introduction  of  the  killed  pathogenic  agent  even  may 
be  equivalent  in  its  effects  to  infection.  That  infection  with  living 
virulent  organisms  may  result  in  protection  has,  of  course,  been  recog- 
nized as  long  as  we  have  had  knowledge  of  the  etiologic  connection 
of  microorganisms  with  the  infectious  diseases,  but  the  discovery 
that  the  same  result  can  be  achieved  in  many  instances  without  the 
production  of  any  malady  of  moment,  through  the  use  of  organisms 
whose  virulence  has  been  artificially  diminished,  and,  as  I  have 
already  indicated,  even  with  organisms  that  are  dead,  is  one  of  the 
greatest  triumphs  of  modern  medicine.  The  various  methods  that 
are  employed  to  this  end  have  already  been  briefly  considered  in  a 
previous  chapter,  and  will  be  taken  up  in  greater  detail  in  connection 
with  the  different  infections  against  which  active  immunization  is 
employed. 

SMALLPOX 

It  is  an  interesting  coincidence  that  the  principle  just  stated,  viz., 
the  possibility  of  producing  active  immunity  by  the  use  of  organisms 
that  have  been  attenuated  in  their  virulence,  was  unconsciously 
employed  by  the  earliest  workers  in  this  field. 

When  and  how  the  discovery  was  made  that  the  virulence  of  small- 


176  ACTIVE  IMMUNIZATION 

pox  is  greatly  diminished  by  the  introduction  of  the  virus  through 
the  skin  is  not  known.  But  the  principle  was  evidently  already  ex- 
tensively utilized  in  Turkey  for  prophylactic  purposes  early  in  the 
eighteenth  century.  For  in  1718  Lady  Montagu,  the  wife  of  the 
English  ambassador  at  the  Ottoman  court,  wrote  to  a  friend  as 
follows:  "The  smallpox  so  fatal  and  general  amongst  us,  is  here 
entirely  harmless  by  the  invention  of  engrafting  which  is  the  term 
they  give  it.  Every  year  thousands  undergo  the  operation,  and  the 
French  ambassador  says,  pleasantly,  that  they  take  the  smallpox 
here  by  diversion,  as  they  take  the  waters  in  other  countries.  There 
is  no  example  of  anyone  who  has  died  in  it,  and  you  may  well  believe 
I  am  satisfied  of  the  safety  of  the  experiment,  since  I  intend  to  try 
it  on  my  dear  little  son.  I  am  patriot  enough  to  take  pains  to  bring 
this  useful  invention  into  fashion  in  England." 

As  a  matter  of  fact  Lady  Montagu's  daughter  was  the  first  person 
inoculated  for  prophylactic  purposes  in  England.  The  material 
used  for  this  purpose  was  the  purulent  matter  obtained  from  small- 
pox pustules  "of  the  distinct  kind,"  which  was  then  applied  to  two 
small  incisions  just  through  the  skin,  on  "Dossils  of  Lint." 

Of  the  subsequent  occurrences,  Dr.  Allen,  a  fellow  of  the  Royal 
Society,  then  gives  the  following  account:  "About  the  eighth  day 
after  the  operation  some  Pustules  begin  to  appear,  not  unlike  to  those 
that  are  commonly  seen  in  the  distinct  kind,  a  little  Fever  having 
preceded  the  Eruption,  and  the  other  usual  Symptoms,  but  more 
mild  and  gentle.  ...  In  the  general  it  is  observable  that  the 
Smallpox  procured  by  Inoculation  are  of  the  distinct  kind,  for  the 
most  part  void  of  danger,  that  the  Pustules  are  few  in  number  and 
pit  very  little."  With  this  method  many  thousands  of  persons  were 
subsequently  treated. 

As  regards  the  prophylactic  value  of  these  inoculations  in  England 
accurate  statistical  reports  are  unfortunately  lacking,  but  it  seems 
from  the  writings  of  contemporary  observers  that  the  protection 
was  regarded  as  complete.  As  regards  the  dangers  of  the  process 
there  is  some  diversity  of  opinion.  The  Sutton  brothers,  who  did  a 
great  deal  to  perfect  the  technique  of  inoculation,  thus  claim  to  have 
inoculated  not  less  than  20,000  persons  without  losing  one  as  the 
direct  result  of  the  operation.  Dr.  Gregory,  of  the  London  Smallpox 
Hospital,  placed  the  mortality  rate  at  one  in  five  hundred.  Sir 
Thomas  Watson  writes:  "No  doubt  the  distemper  was  produced 


SMALLPOX  177 

artificially  in  many  more  persons  than  would  have  caught  it  naturally, 
had  inoculation  never  been  thought  of.  So  that  while  the  relative 
mortality,  i.  e.,  the  percentage  of  deaths  from  smallpox,  was  lessened 
by  this  practice,  the  absolute  mortality  was  fearfully  increased." 

It  was  noticed,  moreover,  that,  contrary  to  expectation,  persons 
who  had  been  variolated  occasionally  themselves  became  centres 
of  infection.  Evidently  the  attenuation  of  the  organism  by  skin 
passage  was  not  always  sufficient  to  make  variolation  an  altogether 
harmless  procedure.  The  underlying  principle,  however,  is  evidently 
sound,  and  for  all  ages  to  come  these  early  attempts  at  protective 
inoculation  will  form  one  of  the  great  turning  points  in  the  history 
of  medicine. 

The  next  step  in  advance  is  intimately  associated  with  the  name  of 
Jenner.  Led  by  the  popular  belief  which  was  prevalent  in  Gloucester- 
shire during  the  latter  half  of  the  eighteenth  century,  that  individuals 
who  have  accidentally  become  infected  with  cowpox  were  thereby 
protected  against  smallpox,  Jenner  actually  put  this  idea  to  the 
test  (1796). 

To  this  end  he  inoculated  a  healthy  boy,  of  eight  years,  with 
material  taken  from  a  cowpox  vesicle  on  the  hand  of  a  dairy  maid,  and 
a  couple  of  months  later  showed  by  inoculation  with  smallpox  virus 
that  the  child  was  actually  immune.  In  1798  he  furnished  further 
proof  that  cowpox  will  furnish  protection  against  smallpox  by 
inoculating,  or,  as  we  may  now  say,  vaccinating  (from  vacca,  the 
cow),  a  child  directly  from  a  cowpox  vesicle,  and  continuing  the 
inoculation  from  arm  to  arm  through  a  series  of  five  children,  after 
which  all  five  were  variolated,  i.  e.,  inoculated  with  human  smallpox, 
the  result  again  being  negative. 

Hereafter  vaccination  was  extensively  practised  in  different 
European  countries,  and  also  introduced  in  America,  the  source  of 
material  for  a  long  time  being  lymph  which  was  obtained  from  cows 
that  had  developed  cowpox,  and  in  some  instances  from  horses, 
affected  with  grease,  the  affections  having  been  shown  to  be  identical. 
While  Jenner  and  many  later  investigators  failed  to  recognize  the 
identity  of  human  smallpox  with  cowpox,  as  well  as  grease,  this 
seems  now  to  have  been  satisfactorily  established,  the  apparent 
differences  between  the  two  conditions  and  the  effect  of  the  inocula- 
tion of  the  two  kinds  of  virus  being  the  result  of  the  attenuation  of 
the  organism  in  question,  in  consequence  of  animal  passage. 
12 


178  ACTIVE  IMMUNIZATION 

While  in  former  years  vaccination  was  frequently  performed  by 
direct  transmission  from  arm  to  arm,  this  has  now  been  entirely 
abandoned,  animal  lymph  being  exclusively  used.  This  is  prepared 
in  special  laboratories,  and  put  up  in  such  form  that  the  practitioner 
can  carry  out  the  vaccination  at  any  place,  whereas  in  former  years 
the  persons  who  were  to  be  vaccinated  were  often  obliged  to  come 
to  the  stables  in  which  the  animals  that  furnished  the  lymph  were 
kept. 

Preparation  of  the  Vaccine. — The  technique  employed  in  the  prepa- 
ration of  the  vaccine  is  in  brief  the  following  (method  in  use  at  the 
Government  Vaccine  Institute  of  Vienna).  The  animals  used  are 
young  cattle,  not  older  than  two  years  or  younger  than  six  months, 
whose  freedom  from  disease  has  been  previously  ascertained.  After 
being  placed  on  the  operating  table  the  abdomen  up  to  the  umbili- 
cus, as  also  the  portion  of  the  inner  surface  of  the  thighs,  is  shaved, 
the  skin  cleansed  with  green  soap  and  water,  then  copiously  rinsed 
with  a  2  per  cent,  lysol  solution,  then  with  sterile  water,  and  finally 
dried  with  sterile  gauze.  The  entire  surface  is  then  scarified  in 
longitudinal  or  transverse  streaks,  measuring  about  10  cm.  in  length, 
and  from  2  to  2.5  cm.  apart,  care  being  taken  that  the  papillary 
layer  is  just  barely  touched,  so  that  there  is  no  bleeding.  The  virus 
is  then  introduced  into  these  streaks,  either  by  making  use  of  a 
special  vaccine  lancet  (Chalybaeus  lancet)  or  by  rubbing  it  in  with 
a  suitable  instrument. 

In  Vienna,  where  so-called  retrovaccination  lymph  is  exclusively 
prepared,  calf  lymph  is  first  inoculated  into  a  healthy  child,  when 
lymph  from  this  source  is  employed  to  inoculate  the  new  animal; 
the  resultant  material  is  termed  retro  vaccine  of  the  first  generation. 
This  can  then  be  used  for  human  vaccination,  or,  still  better,  the 
product  obtained  with  it  from  a  second  animal — the  so-called  retro- 
vaccine  of  the  second  generation. 

After  the  animal  has  been  prepared,  as  just  described,  the  entire 
vaccinated  surface  is  suitably  protected  against  dirt  and  infection, 
and  the  animal  returned  to  its  stable,  which  is  kept  scrupulously 
clean.  The  result  is  seen  in  Fig.  11,  which  represents  the  appearance 
of  the  "pox"  at  the  end  of  five  days.  At  this  time,  or  after  three  to 
four  days  in  younger  animals,  the  covering  is  removed,  the  entire 
surface  cleansed,  as  described  before,  but  not  dried,  when  with  the 
aid  of  a  stout  curette  the  surface  material  is  scraped  off,  care  being 


SMALLPOX 


179 


taken  that  it  is  not  contaminated  with  blood.  With  practice  the 
vesiculated  epithelium  can  be  removed  in  long  strips.  The  material 
is  placed  in  a  suitable  receptacle,  weighed  and  treated  with  five 
times  its  amount  of  sterile  glycerin-water  (80  parts  of  glycerin  and 
20  of  water) .  The  quantity  which  can  usually  be  obtained  from  one 
animal  varies  between  25  and  50  grams  (in  the  case  of  retrovaccine 
of  the  second  generation).  Twenty-four  hours  after  the  removal 
of  the  material  the  animal  is  killed,  and  if  no  disease  that  could 


FIG.  11 


Belly  of  heifer,  showing  one  of  the  approved  modern  methods  of  propagating  vaccine  virus; 
lesions  photographed  at  the  end  of  five  days.     (Taken  from  Welch  and  Schamberg.) 

affect  the  vaccine  is  found  post  mortem,  this  is  further  treated  as 
follows:  After  standing  for  four  weeks  in  contact  with  the  glycerin 
the  mixture  is  thoroughly  triturated  in  a  "lymph  mill,"  when  the 
resultant  emulsion  is  filtered  through  gauze  and  is  then  stored  for 
at  least  three  or  four  weeks  at  a  temperature  of  8°  C.,  the  idea  being 
to  favor  the  destruction  by  the  glycerin  of  contaminating  micro- 
organisms, the  admixture  of  which  is  practically  unavoidable,  even 
though  the  field  of  operation  be  ever  so  carefully  protected. 

If  a  bacteriological  examination  then  shows  the  presence  of  but 


180 


ACTIVE  IMMUNIZATION 


few  (e.  g.,  less  than  30  per  0.01  c.c.)  and  the  absence  of  all  suspicious 
organisms,  including  the  tetanus  bacillus  (the  latter  point  is  tested 
by  injecting  a  mouse  with  0.01  c.c.),  the  lymph  is  placed  in  capillary 
tubes,  which  are  sealed  at  the  ends,  and  is  then  ready  for  use.  As 
the  activity  of  the  vaccine  diminishes  in  the  course  of  time,  each 
preparation  bears  on  its  label  the  date,  after  which  it  should  no 
longer  be  employed. 

Fia.  12 


Vaccine  vesicle  upon  the  seventh  day;  areola  just  beginning.    (Taken  from  Welch  and  Schamberg.) 

The  Process  of  Vaccination. — The  field  of  vaccination,  which  is 
preferably  the  upper  portion  of  the  upper  arm,  is  first  cleansed 
with  soap  and  water,  and  then  with  alcohol  or  ether.  With  a  suit- 
able instrument,  which  must  be  previously  sterilized,  two  or  three 
parallel  scratches  are  then  made,  about  J  cm.  in  length  and  3  cm. 
apart.  A  stout  needle  answers  all  purposes,  and  can  be  sterilized 
by  flaming  the  point  and  then  cooling  it  in  alcohol.  If  desired,  a 
new  needle  can  be  used  for  each  case.  Care  should  be  taken  that 
the  scratch  extends  to,  but  not  through  the  papillary  layer;  actual 
bleeding  is  to  be  avoided.  The  needle  can  either  be  charged  with 
the  lymph  from  the  start,  and  the  scratches  made  with  the  vaccine 
point,  or  a  drop  of  the  material  is  placed  upon  the  scarified  area  and 
subsequently  rubbed  into  the  little  grooves  with  the  body  of  the 
needle.  The  removal  of  the  lymph  from  the  tubes  is  accomplished 


FIG.  13 


Vaccine  vesicle  upon  seventh  day,  showing  beginning  areola.    Patient  was  suffering  from  scarlet 
fever.     Vesicle  shows  some  irregularity  in  form.     (Taken  from  Welch  and  Schamberg.) 


FIG.  14 


Vaccine  vesicle  upon  ninth  day,  showing  more  pronounced  areola.     Same  patient  as  Fig.   13. 
(Taken  from  Welch  and  Schamberg.) 


182  ACTIVE  IMMUNIZATION 

by  the  aid  of  the  tiny  rubber  nipple,  which  is  sent  out  by  most  of 
the  manufacturers  with  each  set  of  tubes.  When  the  vaccination 
is  completed  the  arm  is  usually  left  exposed  until  the  lymph  has 
dried,  so  far  as  this  is  possible  in  the  presence  of  glycerin.  In  Vienna 
it  is  customary  to  cover  each  scarification  with  a  so-called  tegmin 
dressing,  which  may  be  removed  the  following  day  or  the  day  after. 
Subsequently  the  entire  area  may  be  dusted  once  or  twice  a  day 
with  a  powder  composed  of  10  grams  each  of  oxide  of  zinc  and  starch 
and  40  parts  of  talcum.  This,  however,  is  not  necessary. 

The  appearance  of  the  arm  illustrating  the  results  of  a  typical 
vaccination  is  shown  in  the  accompanying  illustrations  (Figs.  12,  13, 
and  14). 

The  Protective  Value  of  Vaccination. — This  is  now  so  generally 
recognized  that  it  scarcely  seems  worth  while  to  enter  into  a  dis- 
cussion of  the  question.  Smallpox,  which  up  to  the  time  of  Jenner 
was  one  of  the  worst  scourges  of  the  civilized  world,  has  now  become 
so  rare  a  disease  in  those  countries  where  vaccination  is  thoroughly 
carried  out  that  the  majority  of  physicians  and  medical  students 
have  not  seen  even  a  single  instance  of  the  disease.  In  Berlin, 
where  the  annual  death-rate  from  smallpox  before  the  introduction 
of  vaccination  varied  between  250  and  400  per  100,000  inhabitants, 
the  aggregate  death-rate  from  the  disease  in  entire  Germany,  even 
including  imported  cases,  is  now  less  than  0.1  per  100,000.  An 
excellent  idea  of  what  systematic  vaccination  can  accomplish  may 
also  be  formed  from  the  accompanying  table,  which  indicates  both 
the  morbidity  and  mortality  from  smallpox  in  the  German  army, 
as  contrasted  with  the  results  in  the  armies  of  Austria,  France, 
and  Italy,  in  which  no  systematic  vaccination  had  been  attempted: 


Morbidity       Average     Mortality    Average 


Army. 

German 
Austrian 
French  . 
Italian  . 


Period. 

(No.  of 
cases). 

per 
year. 

(No.  of 
cases)  . 

per 
year. 

1875  to  1892 

13 

0.7 

1 

0.05 

1875  to  1886 

9864 

896.7 

595 

54.0 

1875  to  1892 

8356 

491.5 

705 

41.4 

1875  to  1894 

2565 

135.0 

193 

10.1 

The  same  point  is  also  well  illustrated  by  comparing  the  number  of 
deaths  during  the  Franco-Prussian  War  in  the  entire  German  army 
—459,  with  that  in  the  French— 24,469. 


RABIES  183 

In  the  face  of  such  evidence  a  country  that  will  not  or  cannot 
enforce  systematic  vaccination  evidently  courts  the  disease,  and 
hardly  merits  a  place  in  the  rank  of  civilized  nations. 

RABIES 

While  the  actual  principle  underlying  the  preventive  vaccina- 
tion against  smallpox  was  scarcely  recognized  by  Jenner  and  his 
contemporaries,  their  work  nevertheless  constitutes  the  basis  of  all 
our  modern  vaccine  work,  and  to  it  may  be  directly  attributed  the 
successful  preventive  treatment  of  another  dreadful  disease,  the 
pathogenic  agent  of  which  has  likewise  not  yet  been  isolated,  viz., 
rabies.  This  discovery  we  owe  to  the  genius  of  Pasteur,  and  to  him 
undoubtedly  belongs  the  credit  for  having  first  recognized  that  by 
the  use  of  suitably  attenuated  virus  full  protection  may  be  afforded 
against  the  corresponding  full-virulent  infection.  In  Jenner's  case 
nature  had  performed  the  experiment  for  him;  but  Pasteur  was  the 
first  who  purposely  employed  the  animal  experiment  to  demonstrate 
the  principle  in  question. 

The  idea  underlying  Pasteur's  antirabic  treatment  is  to  immunize 
the  bitten  individual  within  the  period  of  time  that  the  actual 
disease  requires  for  its  development.  To  accomplish  this  it  was 
necessary  so  to  change  the  nature  of  the  virus  that  the  incubation 
period  following  its  injection  should  be  materially  shorter  than  that 
of  the  actual  disease,  which  is  usually  from  two  to  three  weeks,  but 
may  be  as  long  as  two  months,  or  even  longer. 

This  was  accomplished  by  passing  the  natural  virus,  or  street  virus, 
as  it  is  usually  termed,  through  a  series  of  fifty  rabbits,  when  its 
period  of  incubation  was  found  to  be  reduced  to  but  six  to  eight  days. 
Further  passage  does  not  change  this,  and  such  virus,  which,  more- 
over, no  longer  produces  the  symptoms  of  furious  rabies  in  dogs  or 
guinea-pigs,  but  merely  the  paralytic  type  of  the  disease,  is  now 
termed  virus  fixe.  In  a  certain  sense,  viz.,  insofar  as  the  effect  of 
the  animal  passage  upon  the  period  of  incubation  is  concerned,  we 
may  look  upon  the  virus  fixe  as  being  increased  in  virulence,  but  so 
far  as  its  pathogenic  properties  go  there  is  reason  to  think  that  for 
man  this  is  actually  diminished.  Pasteur  then  found  that  the 
virulence  of  the  virus  in  question  can  be  still  further  diminished  by 
desiccation,  and  that  after  twelve  to  fourteen  days  it  is  lost  alto- 
gether. The  plan  of  treatment  then  is  to  inoculate  the  patient 


184  ACTIVE  IMMUNIZATION 

on  successive  days  with  material  of  increasing  virulence,  beginning 
with  that  which  is  altogether  innocuous,  i.  e.,  twelve  to  fourteen 
days  old. 

The  technique  employed  in  the  preparation  of  the  virus  and  the 
immunization  of  the  patient,  as  described  below,  is  that  in  use  at 
the  Pasteur  Institute  of  the  College  of  Physicians  and  Surgeons  of 
Baltimore,  under  the  direction  of  Dr.  N.  G.  Keirle,  and  represents 
the  original  Pasteur  method. 

Preparation  of  the  Virus. — The  original  virus  was  obtained  from  the 
Pasteur  Institute  of  Paris,  and  had  been  started  from  the  medulla 
of  a  rabid  cow  in  1882.  It  has  since  been  transferred  from  rabbit 
to  rabbit,  and  has  now  reached  about  the  nine  hundredth  remove. 
Another  virus  was  started  by  Dr.  Keirle  himself  from  the  medulla 
of  a  rabid  cow,  and  has  reached  about  the  six  hundredth  remove. 
As  a  rabbit  will  live  about  twelve  days  after  inoculation,  about 
thirty  passages  may  be  made  in  a  year. 

The  inoculations  are  made  as  follows:  "The  hair  between  a  line 
drawn  transversely  through  the  ears  and  eyes  is  cut  off  with  scissors 
and  washed  with  a  3  per  cent,  solution  of  carbolic  acid.  No  anesthetic 
is  required;  the  animal  does  not  cry  out,  and  evinces  no  sign  of  pain. 
The  animal  need  not  be  strapped  down,  but  may  be  held  on  the  table 
by  an  assistant.  A  cut  one  inch  and  a  quarter  long  is  made  with 
the  knife  or  scissors,  longitudinally,  through  the  skin  in  the  middle 
of  the  space  at  the  top  of  the  head  between  the  lines  above  named. 
A  blepharostat  keeps  the  incision  apart,  and  the  sublying  tissue  is 
scraped  away  so  as  to  expose  the  bone  a  little  to  one  side  of  the  median 
line.  The  trephine  has  a  diameter  of  5  mm.  and  a  ring  guard  which 
is  set  at  2  mm.  from  the  cutting  end  of  the  crown;  the  trephine  may 
be  a  bit  fastened  in  a  revolving  drill  handle,  or  a  simple  hand  trephine 
made  of  metal  rod  17  cm.  long.  The  button  of  bone  is  removed  with 
a  tenaculum  and  the  dura  is  exposed,  and  an  ordinary  hypodermic 
syringe  is  used  to  inject  three  or  four  drops  of  the  rabic  emulsion 
beneath  the  dura.  If  the  perforating  end  of  the  needle  is  curved 
almost  at  a  right  angle  for  a  space  of  4  mm.  it  facilitates  its  introduc- 
tion, but  this  is  by  no  means  indispensable.  The  wound  is  closed 
by  interrupted  sutures  (three  are  generally  sufficient)  and  then  sealed 
with  collodion.  The  ordinary  suture  needle  can  be  used,  but  the  risk 
of  sticking  the  hand  is  lessened  if  the  needle  has  a  fixed  handle, 
the  other  extremity  terminating  in  a  spear  with  a  slit  in  its  side, 


RABIES  185 

which  is  opened  and  closed  by  pushing  a  button.  It  is  passed  through 
the  skin  closed,  then  opened,  the  thread  inserted,  when  it  is  again 
closed  and  withdrawn.  Of  course,  all  the  materials  and  instruments 
have  been  sterilized,  and  during  the  operation  are  placed  in  a  pan 
of  3  per  cent,  carbolic  acid.  The  rabbit  is  then  placed  in  a  box  prop- 
erly labelled.  Wire  cages  are  generally  used,  but  if  the  floor  be  of 
asphalt  or  of  cement,  a  box  without  a  bottom,  having  a  wire  grating  for 
a  lid,  with  a  bed  of  sawdust  or  straw,  is  more  convenient  to  keep  clean. 

"In  an  institute  like  the  one  at  Baltimore,  where  approximately 
120  cases  are  treated  annually,  two  animals  are  daily  inoculated, 
the  material  for  this  purpose  being  obtained  from  the  medulla  of 
those  animals  which  have  died  of  rabies  during  the  day  or  the  night 
preceding.  To  this  end  a  piece  from  the  floor  of  the  fourth  ventricle, 
measuring  about  2  cm.,  in  length  is  rubbed  up  in  1  c.c.  of  bouillon, 
and  of  this  emulsion,  as  I  have  just  stated,  three  or  four  drops  are 
injected  beneath  the  dura. 

"As  I  have  mentioned  before,  rabbits  that  have  been  inoculated 
with  virus  fixe  develop  rabies  after  an  incubation  period  of  from 
six  to  eight  days  (the  shortest  period  being  usually  only  reached 
after  ninety  passages),  and  then  die  almost  four  days  later,  viz., 
after  ten  to  twelve  days  following  the  inoculation.  The  dead 
animals,  as  soon  after  death  as  possible,  are  sprayed  with  lysol  or 
bichloride  and  stripped  of  their  fur,  when  the  cord  and  brain  are 
removed  under  aseptic  precautions.  The  cord  is  severed  just  below 
the  medulla  and  divided  into  two  equal  pieces,  which  are  suspended 
by  sterilized  silk  threads  in  a  sterile  glass  jar  (aspiration  or  irrigation 
bottles,  1  liter  capacity),  the  bottom  of  which  has  been  covered 
about  2  cm.  deep  with  flake  caustic  potash.  The  threads  are  held 
in  position  by  the  cotton  stopper  and  are  allowed  to  hang  outside. 
The  medulla  is  kept  in  a  sterile  dish  and  is  used  to  continue  the  series 
of  inoculations,  as  indicated  above.  The  jars  are  labelled  with  the 
date,  the  number  of  the  passage,  and  the  number  of  the  animal 
passage.  A  post  mortem  finally  is  performed  and  any  cord  rejected 
in  which  the  animal  is  found  diseased,  or  in  which  bacteriological 
examination  of  the  cord  has  shown  the  presence  of  pyogenic  organ- 
isms.1 The  jars  are  then  kept  in  the  cord  room  (occupying  about 
12  to  15  square  feet)  at  a  temperature  of  from  20°  to  25°  C. 

1  To  this  end  a  small  piece  is  snipped  off  after  twenty-four  hours,  dropped 
into  bouillon,  and  this  incubated  until  the  next  day. 


186  ACTIVE  IMMUNIZATION 

"In  preparing  the  emulsion  with  which  to  inject  patients  it  is 
better  to  use  the  main  room,  and  have  a  table  with  a  drawer  in  which 
are  labels,  glass  rods,  curved  scissors,  and  forceps,  the  glass  rods 
having  been  wrapped  in  paper  and  sterilized  in  hot  air.  Erlenmeyer 
flasks,  100  c.c.  capacity,  with  cotton  plugs,  contain  water,  sterilized 
in  the  autoclave.  There  are  also  stout  wine-glasses  covered  with 
paper  caps  and  sterilized  in  hot  air;  the  wine-glass  inside  does  not 
taper  to  a  point,  but  has  a  flat  bottom  2  cm.  in  diameter,  which 
corresponds  to  the  diameter  of  the  glass  rods,  which  are  25  cm. 
long;  the  wine-glass  brimful  holds  40  c.c.  There  is,  of  course,  a 
Bunsen  burner  on  the  table. 

"For  the  newly  arrived  patient  the  cord  of  the  thirteenth  and 
fourteenth  day  is  used;  that  is,  a  cord  that  has  been  drying  over 
caustic  potash  for  thirteen  and  fourteen  days.  Such  a  cord  measures 
transversely  J  cm.  The  cord  is  for  one  instant  passed  into  the  Bunsen 
flame,  then  with  curved  scissors,  previously  heated  to  redness  in 
the  flame  and  allowed  to  cool,  less  than  a  millimeter,  is  transversely 
cut  from  the  end  of  the  cord  and  allowed  to  drop  into  the  wine- 
glass; ten  such  pieces  measure  8  mm.  and  weigh  20  mgm.  This  is 
triturated  with  the  glass  rod  until  it  has  become  thoroughly  broken 
or  mashed  according  as  it  is  recent  or  old.  Then  a  few  drops  of 
water  are  added  and  it  is  triturated  until  a  milky  fluid  results,  then 
more  water  added  until  finally  15  c.c.  is  reached. 

"The  emulsion  now  looks  like  rice  water,  and  a  sediment  soon 
accumulates;  the  paper  cap  is  put  back  on  the  wine-glass,  and 
on  the  bottom  rim,  upper  surface,  is  put  a  label  with  No.  14  on 
it,  meaning  a  cord  that  has  dried  fourteen  days  (older  cords,  15-day 
cords,  are  rejected  and  the  bottle  cleaned).  The  caustic  potash, 
after  twice  using,  requires  renewal;  in  twenty-eight  days  it  looks 
like  wet  white  candy.  The  glass  rods  wear  smooth  and  require 
to  have  pieces  cut  with  a  file  and  broken  off,  thus  becoming  sharp 
again.  Similar  to  the  above,  the  thirteenth-day  cord  is  prepared 
in  a  wine-glass.  These  wine-glasses  are  put  in  an  agate-ware  tray 
or  baking  pan  in  which  are  placed  pieces  of  filter  paper,  and  a  bowl 
with  some  3  per  cent,  solution  of  carbolic  acid  and  two  Pravaz 
syringes.  A  list  is  made  out  with  the  names  of  the  patients  and  the 
age  of  the  cords — that  is,  the  number  of  days  they  have  been  drying, 
and  the  day  of  the  month  corresponding,  which  is  the  date  on  the 
bottle,  and  the  dose,  e.  g.: 


RABIES  187 

FOR  JUNE  24,  1898 

Cord.  Date.  Dose. 

George  Williams 10  days        June  14       3  c.c. 

Edward  Cook        .......       5  days        June  19       2  c.c. 

"These  lists  are  made  out  and  put  on  the  table  the  day  before  the 
treatment,  and  are  entered  in  the  case  book,  which  records  the 
circumstances  of  the  patient's  case,  e.  g.,  name,  age,  seat  of  bite, 
number  of  bites,  animal  that  inflicted  bites,  cauterized  or  not,  what 
has  become  of  the  animal,  etc. 

"The  temperature  of  the  cord  room  and  of  the  outside  atmosphere, 
and  the  results  of  the  culture  are  recorded  in  a  book  on  the  table  in 
the  main  room.  We  now  take  the  tray  and  go  to  the  patient. 

Treatment  of  the  Patient. — "The  patient  is  permitted  to  stand, 
sit,  or  lie  down,  as  he  or  she  may  desire.  The  Pravaz  syringes  and 
needles,  which  have  been  filled  with  3  per  cent,  carbolic  acid  solution, 
are  emptied  and  washed  out  with  sterilized  water.  These  syringes, 
holding  3  c.c.,  are  filled  by  thrusting  a  needle  through  the  paper  cap 
of  the  wine-glass;  then  the  abdominal  region  of  the  patient  is  bared 
and  the  site  of  the  injection  (hypochondria,  or  anywhere  on  abdomen), 
avoiding  large  veins,  is  wiped  with  filter  paper  wet  with  3  per  cent, 
carbolic  acid  solution.  Then  the  skin  is  raised  in  a  fold  between  the 
fingers  and  the  needle  is  thrust  well  into  the  subcutaneous  tissue. 
It  is  important  to  avoid  injecting  the  layers  of  the  skin,  which  is 
painful,  and  to  avoid  sites  of  previous  injection.  After  the  injection 
a  piece  of  filter  paper  wet  with  the  carbolic  acid  solution  is  put  on 
the  skin  and  allowed  to  remain  for  a  few  seconds. 

"  We  have  not  modified  the  dose  relative  to  age.  In  our  youngest 
patient,  a  girl  of  two  and  a  half  years  old,  and  an  old  lady,  aged 
eighty-four  years,  the  same  doses  were  given.  At  times  there  are 
redness  and  induration  in  the  connective  tissue,  but  there  has  never 
been  pus,  never  cellulitis  of  the  slighest  gravity.  Hot-water  appli- 
cations on  towels  suffice  to  remove  any  trivial  inconveniences.  The 
treatment  occupies  twenty-one  days  at  least. 

"First  day  (thirteenth-  and  fourteenth-day  cord)  3  c.c.  each  at 
the  same  time,  and  so  on  until  the  sixth-day  cord  is  reached  on  the 
fifth  day;  two  injections  of  the  sixth-day  cord  emulsion  are  given 
in  doses  of  2  c.c.  each  at  the  same  time;  subsequent  injections  are: 
sixth  day  (fifth-day  cord),  2  c.c.;  seventh  day  (fourth-day  cord), 


188  ACTIVE  IMMUNIZATION 

2  c.c.;  eighth  day  (third-day  cord),  1J  c.c.  Injections  are  not  given 
of  cords  earlier  than  the  third  day.  Now  begin  again  with  fifth-day 
cord  and  come  down  to  third-day  cord  inclusive;  these  all  now  being 
2  c.c.  doses. 

"If  it  be  thought  desirable  to  approach  at  first  the  more  virulent 
cords  gradually,  when  the  fifth-day  cord  is  reached  a  fifth-day  cord 
may  be  given  again  as  the  next  day's  injection;  so  also  with  the  fourth- 
day  cord,  but  after  this  reduplication  the  course  of  the  injections 
is  resumed  and  maintained  in  daily  succession,  fifth-day  cord, 
fourth-day  cord,  third-day  cord,  and  over  again  until  the  twenty- 
first  day  has  passed,  the  dose  being  2  c.c.  each  time."  (Keirle.) 

Regarding  the  modus  operandi  of  the  antirabic  vaccination  out 
knowledge  is  as  yet  imperfect,  but  it  appears  that  as  a  result  of 
the  immunizing  process  rabicidal  substances  are  formed  which  are 
capable  of  destroying  the  rabic  virus.  Babes  accordingly  combines 
the  active  immunization  with  the  passive  process,  i.  e.,  the  intro- 
duction of  the  serum  of  immunized  animals,  and  apparently  with 
satisfactory  results.  As  rabicidal  serum  has  no  marked  antitoxic 
properties,  and  as  the  symptoms  of  rabies  are  evidently  toxic  in 
origin,  it  is  clear  that  no  special  benefits  can  be  expected  from  its 
use  when  once  the  disease  has  developed. 

Results. — So  far  as  the  results  of  the  antirabic  treatment  are 
concerned  an  analysis  of  31,330  cases,  in  which  the  existence  of 
rabies  in  the  biting  animal  had  either  been  definitely  established 
or  rendered  highly  probable,  shows  an  average  mortality  of  but 
0.75  per  cent.,  which,  no  doubt,  could  be  still  further  reduced  if  the 
treatment  of  the  bitten  persons  could  always  be  instituted  in  time. 

After  the  disease  has  once  developed,  vaccine  treatment  is,  of 
course,  without  avail;  at  present  we  can  only  hope  that  the  future 
may  yet  teach  us  some  method  by  which  the  disease  when  already 
in  actual  progress  may  yet  be  conquered  and  those  unfortunates 
be  saved  from  their  terrible  sufferings. 


TYPHOID  FEVER  189 


TYPHOID   FEVER 

After  Pasteur  had  shown  that  it  is  possible  to  produce  active 
immunity  in  animals  against  such  diseases  as  chicken  cholera, 
anthrax,  and  swine  plague,  the  thought  naturally  suggested  itself 
that  the  same  should  be  possible  in  the  case  of  some  of  the  organisms 
which  are  pathogenic  for  man.  Attempts  in  this  direction  showed, 
as  a  matter  of  fact,  that  it  is  perfectly  feasible  to  protect  the  common 
laboratory  animals  against  infections  like  typhoid  and  cholera,  and 
that  this  end  can  be  reached  not  only  by  the  use  of  living  cultures, 
but  even  with  the  killed  organisms.  The  latter  discovery  is,  of 
course,  of  the  greatest  importance,  as  it  unquestionably  hastened 
the  application  of  the  findings  in  the  animal  experiment  to  the 
prophylactic  treatment  of  the  human  being.  The  great  question 
naturally  has  been  how  large  a  dose  of  bacilli  should  be  injected  and 
how  frequently  the  injections  should  be  made  in  order  to  secure 
adequate  protection.  Pfeiffer  and  Kolle,  who  were  probably  the 
first  to  attempt  this  in  the  human  being,  thought  that  the  bacterioly- 
tic  content  of  the  serum  might  possibly  be  used  as  an  indicator  in 
this  respect,  while  Wright,  to  whom  we  are  indebted  for  the  actual 
introduction  of  the  method  into  common  use,  once  thought  that 
the  opsonic  content  of  the  blood  might  prove  of  service  in  this  respect. 
Subsequent  studies,  however,  have  shown  that  a  parallel  between  the 
size  of  the  dose,  the  serum  content  of  protective  substances,  and 
the  degree  of  immunity  does  not  exist,  and  we  may  say  that  our 
present  methods  are  essentially  the  outcome  of  actual  trial,  irre- 
spective of  any  special  index. 

Preparation  of  the  Vaccine. — Wright  recommends  that  the  culture 
from  which  the  vaccine  is  to  be  made  should  first  be  brought  to  a 
certain  degree  of  virulence  by  animal  passage,  and  that  its  rate  of 
growth  in  twenty-four  hours  should  yield  from  1000  to  2000  million 
bacilli  per  c.c.  of  bouillon.  The  first  point  can  be  reached  by  passing 
the  organism  through  a  few  guinea-pigs,  while  the  second  has  to  be 
tried  out.  Whether  either  condition  is  really  imperative  may  be 
questioned.  To  my  mind  it  is  more  important  that  the  vaccine 
should  be  polyvalent,  i.  e.,  that  it  should  represent  a  mixture  of 
a  number  of  different  strains.  As  medium  for  growth  ordinary 
1  per  cent,  peptone  broth  is  used,  which  should  be  as  nearly  neutral 


190  ACTIVE  IMMUNIZATION 

as  possible.  Equal  quantities  of  this,  in  suitable  flasks,  are  inocu- 
lated and  kept  at  body  temperature  for  twenty-four  to  forty-eight 
hours,  after  which  they  are  mixed,  care  being  taken  that  the  neck 
and  upper  portion  of  the  mixing  flask  is  not  soiled  by  the  emulsion, 
as  otherwise  some  of  the  organisms  may  escape  destruction  when 
the  mixture  is  sterilized,  which  is  the  next  step  in  the  operation. 
This  is  carried  out  in  a  suitable  water-bath,  at  as  low  a  temperature 
as  possible;  60°  C.  is  the  usual  temperature  for  this  purpose,  though 
some  writers  advocate  52°  C.  Care  should  be  taken  that  the  water 
in  the  bath  stands  at  least  as  high  as  the  culture  in  the  flask,  and 
that  the  temperature  corresponds  to  the  contents  of  the  flask  and 
not  to  the  surrounding  water.  To  this  end  a  second  flask,  filled  with 
water,  is  placed  alongside  the  culture  flask  and  a  thermometer  sus- 
pended in  it.  As  soon  as  the  temperature  reaches  60°  C.,  the  flame 
of  the  water-bath  is  turned  off.  The  flask  remains  in  the  hot  water 
for  ten  to  fifteen  minutes  longer,  and  is  then  removed.  After  this 
the  sterility  of  the  contents  is  tested  by  plating  out  a  given  quantity 
(1  to  10  c.c.,  according  to  the  amount  of  material)  in  agar  or  by 
inoculation  of  broth.  The  content  of  bacteria  is  next  determined 
as  follows: 

Determination  of  the  Number  of  Bacteria  (according  to  Wright).— 
A  capillary  pipette  provided  with  a  rubber  nipple  (Plate  III,  Fig.  d) 
is  marked  with  a  glass  pencil  about  three-quarters  of  an  inch  from 
the  end,  and  is  then  charged  with  one  volume  of  blood  (obtained  by 
puncture  of  the  thumb  near  the  root  of  the  nail),  one  of  the  bacterial 
emulsion  and  three  volumes  of  saline  (0.9  per  cent.),  the  individual 
portions  being  separated  from  one  another  by  little  air-bubbles.1 
Blood  and  bacteria  are  thoroughly  mixed  by  repeatedly  blowing 
the  contents  of  the  capillary  upon  a  slide  and  drawing  them  up 
again  in  solid  column.  Small  drops  are  mounted  on  clean  slides, 
spread  out  like  blood  specimens,  and,  after  drying,  stained  with 
Jenner's  stain  or  one  of  the  numerous  Romanowsky  modifications. 
A  small  square  diaphragm  made  of  paper  or  card-board  is  placed 
in  the  ocular  of  the  microscope,  when  the  red  cells  and  bacteria  are 
counted  in  successive  fields  until  1000  of  the  former  have  been  gone 

1  With  other  bacteria,  where  the  content  may  be  much  smaller  one  may  take 
two  or  three  volumes  of  the  emulsion,  instead  of  saline,  due  allowance  being 
made  in  the  calculation  by  dividing  with  two,  three,  or  four,  as  the  case  may  be. 


PLATE   III 


Apparatus  for  Opsonic  Work. 

(a)  pipette  for  collecting  blood;  (6)  tube  to  receive  blood  for  separation 
of  serum;  (c)  Wright  blood  capsule;  (d)  blood  pipette  charged  with  cor- 
puscles, serum,  and  bacterial  emulsion;  (e)  same  in  solid  column,  ready 
for  incubation. 


TYPHOID  FEVER 


191 


FIG.  15 


CC 

— 10 


over.  As  the  number  of  red  cells  in  1  c.c.  of  normal  blood  is  about 
5,000,000,000,  and  as  the  red  cells  and  bacteria  must  be  present 
in  the  mixture  in  the  same  ratio  to  one  another  as  in  the  original 
units  of  volume,  the  number  of  bacteria  per  cubic  centimeter  of  the 
vaccine  is  ascertained  according  to  the  equation:  Number  of  red 
cells  counted  :  number  of  bacteria  counted  :  :  5,000,000,000  :  x.  This 
method,  of  course,  has  no  claims  to  accuracy,  but  it  is  the  one 
which  is  usually  employed  in  titrating  out  vaccines.  A  more 
accurate  method  has  of  late  been  suggested  by  Hopkins,  which 
seems  to  have  many  points  in  its  favor. 

Hopkins'  Method.1 — This  is  based  upon 
the  concentration  of  a  bacterial  culture 
by  centrifugation  and  the  preparation  of 
standard  emulsions  from  the  sediment. 
To  this  end  the  washings  from  slant 
cultures,  after  filtration  through  a  small 
cotton  filter  to  remove  larger  clumps  of 
bacteria  and  particles  of  agar,  are  placed 
in  especially  constructed  centrifugation 
tubes  (prepared  by  the  International 
Instrument  Company  of  Cambridge, 
Mass.,  see  Fig.  15),  covered  with  rubber 
caps,  and  centrifugalized  for  half  an 
hour  at  a  speed  of  approximately  2800 
revolutions  per  minute.  The  salt  solu- 
tion and  bacteria  above  the  0.05  mark 
are  then  removed  and  5  c.c.  of  saline 
solution  measured  into  the  tube,  so  as  to  make  a  1  per  cent, 
emulsion.  If  the  sediment  does  not  reach  the  0.05  mark  its 
volume  is  read  on  the  scale  and  a  corresponding  quantity  of 
saline  added  to  make  the  emulsion  1  per  cent,  in  strength.  By 
means  of  a  capillary  pipette  armed  with  a  nipple  the  organisms  are 
forced  into  suspension,  when  the  vaccine  is  transferred  to  a  tube, 
to  be  killed  in  the  usual  manner. 

Estimations  of  carefully  counted  suspensions  obtained  by  centrifu- 
gation in  the  above  manner  gave  the  following  results: 


Special  centrifuge  tube  with 
graduated  tip. 


1  Jour.  Amer.  Med.  Assoc.,  vol.  xl,  No.  21,  p.  1615. 


192  ACTIVE  IMMUNIZATION 

Per  cent.  Billion  per  c.c. 

Staphylococcus  aureus  and  albus     ...  1  10.0 

Streptococcus  hemolyticus 1  8.0 

Gonococcus 1  8.0 

Pneumococcus 1  2.5 

Bacillus  typhosus 1  =               8.0 

Bacillus  coli 1  4.0 

With  different  speeds  different  standards  will,  of  course,  be 
obtained. 

After  the  strength  of  the  emulsion  has  been  determined  the 
material  is  diluted  to  the  desired  degree  with  carbolic  acid,  such  that 
the  final  content  of  the  latter  shall  be  between  0.25  and  0.5  per  cent. 
This  then  constitutes  the  vaccine  and  keeps  practically  indefinitely. 
The  manufacturers  now  furnish  this  in  little  ampoules  containing  the 
requisite  dose. 

Dose  and  Method  of  Vaccination. — For  practical  purposes  Wright 
recommends  a  first  injection  of  750,000,000  to  1,000,000,000  organ- 
isms, and  double  this  dose  for  the  second  treatment.  These  injec- 
tions may  be  made  practically  anywhere,  where  the  skin  is  not  bound 
down  tightly.  I  have  thus  found  the  outer  aspect  of  the  upper  arm, 
where  the  skin  lies  quite  loose,  a  favorable  locality.  Others  inject 
in  the  loin,  or  in  the  back,  in  the  neighborhood  of  the  shoulder-blade. 
The  injections  are,  of  course,  to  be  made  with  a  sterile  syringe,  and 
after  having  cleansed  the  area  to  be  injected  with  soap  and  water 
and  alcohol,  or,  as  has  recently  been  recommended,  after  painting 
with  tincture  of  iodin.  The  needle  puncture  is  covered  with  collodion. 

Fearing  that  the  injection  of  a  large  dose  of  organisms  may  at 
first  be  followed  by  a  diminution  in  the  protective  substances  of  the 
body  (negative  phase),  owing  to  an  interaction  between  the  normal 
antibacterial  substances  and  the  bacterial  antigen,  and  that  the  indi- 
vidual may  thus  be  temporarily  less  resistant  to  the  corresponding 
infection,  Wright  has  suggested  that  in  persons  who  are  likely  to  be 
exposed  to  typhoid  infection  soon  after  the  first  injection,  this  should 
be  smaller  than  usual,  and  that  its  effect  is  to  be  supplemented  later 
by  a  correspondingly  stronger  injection.  Whether  or  not  such  a 
danger  actually  exists  in  the  case  of  the  human  being  has  not  yet 
been  demonstrated.  In  the  animal  experiments,  such  a  period  of 
diminished  resistance  apparently  does  not  develop,  for  Pfeiffer  and 
Friedberger  have  shown  that  guinea-pigs  which  have  been  vaccinated 


TYPHOID  FEVER  193 

with  large  doses  manifest  an  increased  resistance  as  early  as  eight 
to  thirty-six  hours  following  the  injection. 

Symptoms  following  Vaccination. — Usually  within  two  or  three 
hours  following  vaccination  a  local  reaction  appears  about  the  site 
of  inoculation,  which  is  characterized  by  marked  redness  and  infil- 
tration. This  is  followed  by  elevation  of  temperature  (up  to  102° 
F.),  and  occasionally  by  headache,  general  lassitude,  muscle  pain,  and 
frequently  by  swelling  of  the  regional  lymph  glands.  These  symp- 
toms persist  usually  for  one  to  three  days  and  then  disappear.  In- 
dividually there  is  a  good  deal  of  variation,  both  in  the  extent  of  the 
local  reaction  and  in  the  intensity  of  the  general  symptoms.  The 
majority  of  people  are  very  little  inconvenienced,  and  are  able  to 
continue  about  their  work  as  usual.  In  isolated  cases,  however, 
the  person  may  feel  quite  ill  for  a  number  of  days,  and  suffer  a  good 
deal  of  pain  in  the  arm,  which  may  be  red  and  swollen  over  a  consid- 
erable area.  Prompt  recovery  takes  place  in  every  instance.  For 
the  local  pain,  Wright  recommends  warm  applications  and  a  salve 
composed  of  carbolic  acid,  1.0;  fld.  ext.  of  ergot,  4.0;  oxide  of  zinc,  3.0; 
lanolin,  20.0;  for  internal  use  he  recommends  2.0  grams  of  calcium 
chloride  or  calcium  lactate.  All  observers  remark  that  in  malarial 
cases  the  reaction  is  unusually  severe,  and  had  better  be  avoided. 
The  symptoms  occurring  after  the  second  injection  are  usually 
milder;  Leishman  noted  marked  sweating  during  the  second  night 
following  the  reinjection.  During  the  twenty-four  hours  following 
the  inoculation  the  individuals  should  be  told  to  abstain  from  the  use 
of  alcohol,  and  to  avoid  muscular  exertion  and  exposure  to  the  sun. 

Following  the  inoculations,  there  is  a  marked  increase  in  the 
bactericidal  substances  and  bacteriolysins  of  the  blood,  which 
reaches  its  highest  point  on  the  third  day  following  the  reinoculation 
(fourteenth  day)  in  the  case  of  the  first,  and  on  the  seventh  day 
(eighteenth  day)  in  the  case  of  the  latter.  Generally  speaking  the 
increase  amounts  to  from  five  to  ten  times  the  original  quantity. 
Agglutinin  formation  begins  on  the  ninth  day,  drops  after  the  second 
injection,  and  starts  again  nine  days  later,  reaching  its  maximum 
between  the  twenty-second  and  twenty-fifth  day  (2000  to  4000). 
Normal  opsonins,  according  to  Leishman,  are  not  demonstrable, 
while  the  stimulins  (immune  opsonins,  i.  e.,  elements  occurring  in 
heated  serum,  which  favor  phagocytosis)  are  increased  after  eleven 
days. 
13 


194  ACTIVE  IMMUNIZATION 

The  Duration  of  the  Protection. — The  duration  of  the  protection 
afforded  by  the  vaccination  Wright  estimates  at  from  two  to  three 
years,  while  Kuhn  speaks  of  a  single  year. 

Results. — In  the  human  being  it  is,  of  course,  out  of  the  question 
to  study  the  protective  value  of  the  vaccination,  as  is  possible  in 
the  animal  experiment.  All  that  we  can  do  is  to  compare  the  rate 
of  morbidity  from  typhoid  fever  in  a  large  body  of  vaccinated  indi- 
viduals who  have  been  more  or  less  exposed  to  infection,  with  that 
occurring  in  a  similar  body  of  men  who  have  not  been  protected, 
and  who  have  been  exposed  to  a  similar  extent.  We  can  further 
compare  the  rate  of  mortality  among  the  non-vaccinated  with  that 
of  those  who  have  been  vaccinated,  but  who  have  nevertheless 
developed  the  disease.  Studies  of  this  kind  have  been  carried  on 
in  the  English  army  at  the  time  of  the  Boer  War,  in  the  German 
army  in  Southwest  Africa,  and  lately  in  the  United  States  concen- 
tration camp  on  the  Mexican  border  (1911). 

Some  of  the  data  obtained  in  the  English  army  are  given  in  the 
accompanying  table,  from  which  it  is  clear  that  the  vaccinated 
individual  enjoyed  a  much  greater  security,  both  as  regards  the 
probability  of  infection,  and  the  outcome,  in  the  event  that  the 
disease  nevertheless  developed: 

Non-vaccinated.  Vaccinated. 

Men.         Morbidity.    Mortality.    Men.    Morbidity.    Mortality t 

Indian  Army  (1899)  .      .    25,851         657  146       4502  44               9 

(2.54%)  (0.56%)  (0.89%)    (0.2  %) 
Garrison   of   Ladysmith 

(1899  to  1900)       .      .    10,529       1489  329        1705  35               8 

(14.14%)  (3.12%)  (2.05%)    (0.47%) 
Army    in     Egypt     and 

Cyprus       ....      2,669          68  10       720  1                1 

(2.55%)  (0.37%)  (0.14%)    (0.14%) 
Hospital    at    Bloemfon- 

tain       178  24        53               3 

(14%)  (5.6  %) 

From  this  table  it  is  also  clear  that  the  protection  is  not  absolute. 

The  results  obtained  in  our  own  army  are  even  more  striking. 
It  will  be  recalled  that  both  the  morbidity  and  mortality  from 
typhoid  fever  in  our  concentration  camps  at  the  time  of  the  Spanish 
war  were  perfectly  appalling.  In  a  body  of  10,759  men  there  were 
thus  1729  cases  of  certain  typhoid,  and  in  addition  964  cases  of 


CHOLERA  195 

probable  typhoid  (2693  in  all),  with  248  deaths.  Translated  into 
percentages  this  means  that  of  the  entire  body  of  soldiers  25  per 
cent,  were  taken  ill  with  fever,  which  either  was  definitely  recognized 
or  suspected  as  being  typhoid,  with  a  death-rate  of  9.2  per  cent. 
All  these  men  were  non- vaccinated.  Compare  with  this  the  fact 
that  among  the  12,801  men  who  were  concentrated  in  1911  at  the 
maneuver  camp  at  San  Antonio,  all  of  whom  had  been  vaccinated 
either  before  their  arrival  or  as  soon  thereafter  as  possible,  there 
developed  but  a  single  very  mild  case,  a  private  who  had  not  com- 
pleted his  immunization,  thus  giving  a  percentage  of  0.008 !  During 
the  same  period  there  were  reported  in  the  city  of  San  Antonio  forty- 
nine  cases  and  nineteen  deaths. 

On  the  basis  of  these  findings  it  would  appear  that  with  modern 
sanitary  methods,  coupled  with  vaccination  in  the  case  of  those  who 
are  likely  to  be  exposed  to  infection,  typhoid  fever  should  ere  long 
become  as  rare  in  our  hospitals  as  are  smallpox  and  cholera  at  the 
present  time. 

Antityphoid  Vaccination  for  Curative  Purposes. — Since  the  introduc- 
tion of  antityphoid  vaccination  for  prophylactic  purposes  various 
attempts  have  been  made  to  influence  the  course  of  the  malady 
also  by  such  measures.  Some  writers  indeed  express  themselves 
quite  favorably  on  this  point,  but  it  will  no  doubt  require  a  great 
deal  of  investigation  before  we  can  come  to  any  definite  conclusions. 
It  should  be  remembered  that  we  have  no  indicator  to  tell  us  how 
much  to  inject  and  when  to  inject,  and  aside  from  the  actual  course 
of  the  malady,  which  varies  so  greatly  in  different  cases,  we  have  no 
way  of  knowing  whether  we  are  producing  an  effect  at  all,  let  alone 
whether  this  is  beneficial  or  otherwise.  Whether  or  not  Wright's 
opsonic  index  might  yet  serve  some  purpose  in  the  study  of  such 
cases  the  future  will  have  to  show. 


CHOLERA 

Prophylactic  vaccination  against  Asiatic  cholera  was  first  attempted 
in  1885  by  Ferron,  during  an  epidemic  occurring  in  Spain.  As 
infection  of  the  human  being  with  the  organism  in  question  can  only 
take  place  by  way  of  the  intestinal  canal,  Ferron  injected  living 
organisms  subcutaneously,  using  eight  drops  of  a  broth  culture  for 


196  ACTIVE  IMMUNIZATION 

the  first,  and  0.5  c.c.  for  the  second  and  third,  the  inoculations  being 
made  six  to  eight  days  apart.  His  statistics  and  experiments  on 
guinea-pigs,  which  formed  the  basis  of  his  work  on  the  human  being, 
have  been  adversely  criticised  by  a  number  of  subsequent  investi- 
gators; but  the  fact  remains  that  he  was  the  first  to  attempt  anti- 
cholera  vaccination  in  the  human  being,  that  he  injected  a  large 
number  of  people  (200,000,  according  to  his  statements)  with  living 
cultures,  and  that  his  method  is  essentially  the  same  as  that  which 
Haffkine  subsequently  used  in  India,  and  which  unquestionably  can 
afford  protection.  We  also  know  that  as  a  consequence  of  such  in- 
jections bactericidal  substances  appear  in  the  serum  in  large  amounts, 
and  that  attempts  in  this  direction  hence  have  a  proper  theoretical 
basis. 

The  essential  difference  between  Ferron  and  Haffkine  is  that  the 
latter  uses  an  attenuated  culture  (vaccine  I)  for  his  first  injection, 
and  then  follows  this  with  one  that  has  been  brought  to  a  high  degree 
of  virulence  by  animal  passage,  and  which  in  conformity  with  Pas- 
teur's nomenclature,  he  speaks  of  as  virus  fixe  (vaccine  II).  Later 
investigations  have  shown,  as  a  matter  of  fact,  that  a  high  degree  of 
virulence  is  essential  to  effect  successful  immunization.  But,  like 
Ferron,  Haffkine  thought  it  imperative  to  use  living  cultures.  That 
this  is  unnecessary,  however,  was  subsequently  shown  by  Kolle, 
and  the  results  which  have  thus  far  been  obtained  with  the  latter's 
method,  both  in  the  human  being  and  in  the  animal  experiment, 
seem  to  render  future  work  with  living  cultures  unnecessary  and 
perhaps  even  undesirable. 

Kolle's  Method. — The  vaccine  is  prepared  by  emulsifying  twenty- 
four-hour-old  cultures  of  a  virulent  strain  (increased  by  passage 
through  guinea-pigs)  of  the  cholera  vibrio  in  normal  salt  solution, 
such  that  1.  c.c.  shall  contain  1  oese  (=2  mgrms.)  of  organisms. 
These  are  then  killed  by  exposure  to  60°  C.  for  one-half  hour,  when 
carbolic  acid  is  added,  to  the  extent  of  0.5  per  cent.,  as  preservative. 
Two  injections  are  given  hypodermically  about  a  week  apart,  1  c.c. 
the  first  time  and  2  c.c.  the  second  time.  Care  should  be  taken 
not  to  inject  at  a  place  where  the  skin  is  bound  tightly  down.  Suit- 
able districts  are  the  area  over  the  triceps  and  the  loin. 

Symptoms  following  the  Inoculation. — As  in  the  case  of  the  anti- 
typhoid injections,  the  symptoms  vary  in  different  people.  Locally 
there  is  more  or  less  pain  which  begins  after  five  to  six  hours,  with 


PLAGUE  197 

relatively  little  redness  and  swelling.  There  is  usually  some  eleva- 
tion of  temperature  (101°  to  102°  F.),  headache,  and  general  malaise; 
in  women  nausea  and  vomiting,  and  in  about  10  per  cent,  of  the  people 
diarrhea  on  the  following  day.  After  twenty-four  to  seventy-two 
hours  the  symptoms  have  disappeared. 

Results. — Kolle's  method  has  been  tested  in  Japan  (1902),  and 
has  apparently  furnished  reasonably  satisfactory  results,  even  though 
the  vaccination,  as  in  the  case  of  the  antityphoid  treatment,  does 
not  afford  protection  in  all  cases.  In  a  certain  district  occupied  by 
903,194  people,  Murata  vaccinated  77,907  individuals,  the  result 
being  that  the  morbidity  among  the  latter  was  only  0.06  per  cent., 
as  contrasted  with  0.13  per  cent.,  when  compared  with  the  total 
population,  and  the  mortality  (calculated  in  relation  to  the  mor- 
bidity) only  42.5  per  cent.,  as  compared  with  75  per  cent.  In  actual 
figures  this  means  that  of  825,287  non-vaccinated  people  1152  people 
contracted  the  disease,  resulting  in  863  deaths,  while  of  77,907 
vaccinated  individuals  only  48  were  taken  ill  and  20  died. 

Even  more  convincing  than  these  figures  are  certain  individual 
observations.  In  two  villages  which  were  close  to  a  large  cholera 
focus,  and  in  which  all  the  inhabitants  had  been  vaccinated,  not  a 
single  individual  was  taken  ill,  notwithstanding  a  most  active  inter- 
course between  the  people. 

In  a  branch  office  of  the  Formosa  Camphor  Company  all  but 
three  individuals  were  inoculated  (159).  But  one  of  the  total 
number,  and  this  one  a  non-vaccinated  person,  developed  the 
disease  and  died. 

Similar  results  have  been  obtained  by  Haffkine  in  India, 
so  that  the  conclusion  seems  justifiable  that  vaccination  with 
suitable  material  actually  affords  a  considerable  degree  of  pro- 
tection against  Asiatic  cholera,  and  should  be  enforced  as  far  as 
possible  in  times  of  epidemic.  Coupled  with  modern  sanitary 
methods,  vaccination  should  certainly  remove  a  great  deal  of  the 
danger  which  attaches  to  this  relic  of  medieval  lack  of  civilization. 


PLAGUE 

Attempts  at  prophylactic  vaccination  against  plague  have  likewise 
led  to  encouraging  results.    Haffkine,  who  has  done  a  great  deal  of 


198  ACTIVE  IMMUNIZATION 

the  pioneer  work  in  this  direction,  thus  gives  some  very  convincing 
figures:  In  the  city  of  Hubli  (British  India),  numbering  about 
47,427  inhabitants,  vaccination  was  begun  on  the  llth  of  May, 
1898.  From  this  date  until  the  27th  of  September  38,712  individuals 
had  been  vaccinated,  and  of  these  339,  i.  e.,  0.8  per  cent.,  died. 
Of  the  non-vaccinated  during  the  same  period  2395  succumbed, 
i.  e.,  5  per  cent.,  as  compared  with  the  total  number  of  inhabitants. 
During  the  week  of  September  21  to  September  27,  when  all  the 
inhabitants,  with  the  exception  of  603,  had  finally  been  vaccinated, 
there  were  among  the  38,712  protected  individuals  only  20  deaths, 
while  of  the  603  non-vaccinated  persons  58,  i.  e.,  9.61  per  cent., 
died. 

Quite  striking  also  are  the  following  data:  In  three  villages  there 
occurred  13  cases  among  365  vaccinated  persons,  with  3  deaths, 
while  of  the  363  non-inoculated  individuals  49  were  taken  ill  and  38 
died.  In  Bombay  there  developed  18  cases  of  the  disease  among 
8200  vaccinated  persons,  with  2  deaths  (mortality  11.1  per  cent.), 
while  the  general  mortality  from  the  disease  was  over  90  per  cent. 

These  few  examples  will,  I  think,  suffice  to  illustrate  the  real  value 
of  vaccination  against  plague,  but  it  will  be  noted,  as  in  anticholera 
vaccination,  that  the  protection  is  not  absolute.  The  mortality 
among  the  vaccinated  is  so  much  lower,  however,  i.  e.,  11  to  41 
per  cent.,  as  compared  with  50  to  92  per  cent.,  among  the  non- 
vaccinated,  as  observed  in  different  localities,  that  this  factor  in 
itself  would  establish  the  value  of  the  procedure,  and  as  a  matter  of  fact 
all  the  different  commissions,  which  have  investigated  the  Haffkine 
method,  have  expressed  themselves  in  this  sense. 

The  Duration  of  Protection. — This  is  estimated  at  several  months, 
after  which  the  vaccination  must  be  repeated,  if  danger  of  infection 
still  exists. 

Preparation  of  the  Vaccine.  (Haffkine). — Haffkine  makes  use  of 
bouillon  cultures  which  have  been  allowed  to  grow  for  six  weeks  at  a 
temperature  of  25  to  30°  C.  The  bouillon  is  prepared  as  follows: 
1000  grams  of  lean  (goat)  meat  are  passed  through  a  meat-hashing 
machine,  and  are  digested  for  three  hours  with  125  grams  of  hydro- 
chloric acid  (concent.)  in  the  autoclave,  at  a  pressure  of  three  atmos- 
pheres. The  resultant  material  is  filtered  and  diluted  with  water, 
so  that  the  content  in  proteins  shall  be  1  per  cent,  (approximately 
seven  volumes  of  water).  The  broth  is  then  neutralized  with 


PLAGUE  199 

calcium  carbonate,  and  sodium  chloride  added  to  make  it  of  physio- 
logical strength.  It  is  then  sterilized  and  filtered  into  suitable  flasks 
(to  a  height  of  7.5  cm.),  into  each  of  which  a  few  drops  of  olive  oil  or 
butter  fat  (sterile)  are  further  added,  to  serve  as  floats  from  which 
surface  growth  of  the  bacilli  can  take  place.  Every  two  or  three 
days  the  cultures  should  be  shaken,  so  that  new  crops  of  the  organism 
can  develop  in  contact  with  the  air,  the  older  ones  going  to  the 
bottom. 

After  a  six  weeks'  growth  has  been  obtained,  the  purity  of  the 
culture  is  examined  by  plating  out  a  small  amount  on  agar.  The 
material  is  sterilized  for  one  or  more  hours  at  65°  C.,  treated  with 
carbolic  acid  to  the  point  of  0.5  per  cent.,  and  finally  filled  into  small 
vials  of  30  c.c.  capacity. 

Dose. — The  ordinary  dose  for  adult  males  is  3  to  3.5  c.c.;  for  adult 
females,  2  to  2.5  c.c.;  for  children  more  than  ten  years  old,  1  c.c., 
and  for  smaller  children,  0.1  to  0.5  c.c.  Much  larger  quantities, 
however,  can  be  employed  without  harm,  and  Haffkine  himself 
has  injected  as  much  as  20  c.c.  A  second  injection  is  recommended 
after  eight  to  ten  days.  The  injections  are  made  subcutaneously, 
with  a  sterile  syringe,  into  the  upper  arm  (area  over  the  triceps) 
or  the  loin,  those  districts  being  avoided,  as  usual,  in  which  the  skin 
is  tightly  bound  down. 

Symptoms  following  Injection. — The  symptoms  following  the 
injection  are  practically  the  same  as  those  occurring  after  an  anti- 
cholera  or  antityphoid  vaccination,  and  differ  considerably  in  their 
severity  in  different  people.  After  twenty-four  to  forty-eight  hours 
the  individuals  are  usually  no  longer  inconvenienced. 

After  the  disease  has  once  developed  vaccination  is  of  no  avail, 
and  in  such  cases  serum  treatment  should  be  resorted  to  (see  below) . 
The  combined  procedure  would  suggest  itself  as  being  of  value  when 
there  is  reason  to  think  that  the  person  may  have  already  been 
exposed  to  the  infection  or  when  great  danger  actually  exists.  For 
such  purposes  Shiga  advocates  an  initial  treatment  of  0.6  to  1  c.c. 
each,  of  antiplague  serum  and  vaccine,  which  is  to  be  followed  after 
a  few  days,  i.  e.,  after  the  reaction  has  disappeared,  by  a  second 
injection  of  vaccine  alone.  The  latter  is  essentially  an  emulsion  of 
three-day-old  agar  cultures  (incubated  at  30°  C.),  1  c.c.  of  physio- 
logical salt  solution  being  used  for  each  oese  of  the  culture.  The 
emulsion  is  kept  for  30  minutes  at  60°  C.,  treated  with  carbolic  acid 


200  ACTIVE  IMMUNIZATION 

to  the  extent  of  0.5  per  cent.,  and  allowed  to  stand  for  twenty-four 
hours  before  being  used. 

During  epidemics  Shiga  recommends  that  still  larger  doses  of 
the  vaccine  be  used,  or  to  vaccinate  three  times  with  increasing 
quantities. 

DYSENTERY 

Protective  vaccination  against  bacillary  dysentery  has  likewise 
been  attempted,  but  has  not  as  yet  led  to  results  which  are  compar- 
able in  value  to  what  we  see  in  the  case  of  plague  and  cholera.  Shiga 
himself  inoculated  some  10,000  individuals  between  1898  and  1900, 
and  thought  that  he  could  note  a  decrease  in  the  mortality,  while 
the  morbidity  and  the  severity  of  the  symptoms  were  apparently 
uninfluenced.  The  dead  cultures,  however,  are  so  highly  toxic 
that  this  element  in  itself  is  an  obstacle  to  the  more  general  use  of 
such  a  vaccine.  Whether  further  investigations  in  this  direction 
will  lead  to  more  practical  results  time  only  can  tell,  but  it  would  not 
seem  to  be  out  of  the  question,  particularly  when  coupled  with  the 
use  of  a  corresponding  antitoxic  serum. 

OTHER   DISEASES 

In  the  other  infectious  maladies  to  which  the  human  being  is 
subject  protective  vaccination  has  either  not  yet  been  attempted  or 
has  not  yielded  encouraging  results.  There  are  a  number  of  infec- 
tious diseases,  however,  occurring  in  the  domesticated  animals 
against  which  vaccination  may  be  successfully  employed.  This  is 
notably  the  case  with  anthrax,  swine  plague  (Schweinerotlauf), 
cattle  plague  (Rinderpest),  sympathetic  anthrax  (Rauschbrand), 
and  within  certain  limitations  also  with  cattle  tuberculosis.  For  a 
consideration  of  the  methods  employed  and  the  results  which  have 
been  reached  in  these  diseases  which  so  closely  affect  the  human 
being  the  reader  is  referred  to  special  works. 

(B)    ACTIVE    IMMUNIZATION    FOR    THERAPEUTIC    PURPOSES 

While  in  the  diseases  which  have  thus  far  been  considered,  active 
immunization  will  only  furnish  results  of  value  when  carried  out  for 
prophylactic  purposes,  whereas  the  same  measures  have  no  influence, 


ACTIVE  IMMUNIZATION  FOR  THERAPEUTIC  PURPOSES     201 

so  far  as  we  know,  upon  the  disease,  when  this  has  once  been  estab- 
lished, it  appears  from  recent  studies  that  vaccination  may  advan- 
tageously be  employed  as  a  curative  agent,  in  those  infections  which 
are  characterized  by  a  tendency  to  chronicity,  and  in  which  toxins 
play  little  or  no  role.  The  credit  for  having  established  this  possi- 
bility, and  for  its  popularization,  undoubtedly  belongs  to  Wright. 

Wright's  concept  of  the  rationale  underlying  the  tedious  course 
of  some  of  these  infections  is  essentially  based  upon  the  supposition 
that  the  autovaccinations  which  take  place  in  the  body  of  the  infected 
individual  are  imperfectly  interspaced  as  regards  point  of  time 
and  improperly  adjusted  as  regards  dosage,  the  consequence  being 
that  the  formation  of  certain  protective  substances,  and  notably  of 
the  opsonins,  takes  place  irregularly  and  insufficiently.  He  expressed 
the  opinion  that  by  following  the  opsonic  curve  indications  might 
be  obtained  for  the  introduction  of  the  corresponding  organisms 
from  without  as  vaccines,  both  as  regards  the  size  of  the  dose  and 
the  frequency  of  the  injections,  and  that  it  might  thus  be  possible 
to  favorably  influence  such  infections  as  acne,  sycosis,  furunculosis, 
endocarditis,  chronic  cystitis,  pyelitis,  tuberculosis,  etc.  For  a 
consideration  of  the  details  underlying  Wright's  opsonic  studies,  I 
must  refer  the  reader  to  Wright's  own  publications,  and  the  chapter 
on  the  opsonins  in  the  first  part  of  the  present  work.  Suffice  it  to 
state  at  this  place  that  the  opsonic  index  unfortunately  did  not  fulfil 
those  expectations  with  which  it  was  at  first  greeted,  and  that  any 
attempts  at  vaccine  treatment  must  still  be  made  upon  a  more  or 
less  empirical  basis,  and  with  no  more  definite  or  accurate  index 
to  dosage  and  frequency  of  injection  than  is  afforded  by  the  clinical 
symptoms.  But  even  so,  there  can  be  no  doubt  that  a  certain 
amount  of  good  may  be  accomplished;  how  much,  it  is  yet  impossible 
to  say.  So  much  depends  upon  the  individual  case,  coincidence, 
the  personal  factor  in  the  observer,  etc.,  that  conclusions  should 
only  be  drawn  with  great  care.  As  yet  we  certainly  do  not  know 
enough  of  what  may  or  what  may  not  be  accomplished  to  warrant 
any  dogmatic  statements. 

Preparation  of  the  Vaccines. — A  great  deal  of  discussion  has  arisen 
regarding  the  question  whether  or  not  it  is  imperative  to  use  auto- 
genous vaccines,  i.  e.,  vaccines  that  are  derived  from  the  individual 
organism  which  is  responsible  for  the  particular  infection,  or  whether 
it  is  permissible  to  make  use  of  stock  vaccines,  which  may  in  turn  be 


202  ACTIVE  IMMUNIZATION 

polyvalent,  i.  e.,  composed  of  organisms  derived  from  a  number  of 
cases  of  the  kind  that  is  under  consideration,  or  from  one  single 
case,  but  not  from  the  individual  who  is  to  be  treated.  As  long 
as  we  know  so  little  of  what  vaccines  may  accomplish  it  is  clear 
that  our  clinical  knowledge  is  not  sufficient  to  decide  such  a  question. 
We  can  only  speak  theoretically,  and  theoretically  we  must  admit 
the  probable  existence  of  many  strains  of  a  given  type  of  organism, 
and  with  this  the  possibility  of  individual  differences,  so  that  upon 
this  basis  autogenous  vaccines  would,  cceteris  paribus,  appear  to  be 
preferable  to  stock  vaccines.  But  as  it  is  frequently  and  in  some 
infections  indeed  uniformly  impossible  to  prepare  an  autogenous 
vaccine,  we  may  be  forced  to  use  stock  material  in  many  cases. 

The  preparation  of  the  majority  of  vaccines  is  conducted  as  follows  : 
Cultures  are  made  from  whatever  source  is  available,  i.  e.,  from  the 
pus  of  abscesses,  from  acne  pustules,  the  urine,  blood,  sputum,  etc., 
the  culture  medium  being  chosen  in  accordance  with  the  type  of 
organism  that  is  expected,  or  which  a  preliminary  examination  has 
shown  to  be  present.  If  only  one  type  is  found,  the  vaccine  is 
made  from  it,  while  in  the  event  of  a  multiple  infection,  or  in  the 
presence  of  contaminating  saprophytic  organisms,  the  predominating 
pathogenic  varieties  are  chosen,  i.  e.,  those  which  are  recognized  as 
being  pathogenic  either  by  direct  examination  or  by  passage  through 
an  animal.1 

Having  once  secured  an  initial  supply,  it  is  then  only  necessary 
to  inoculate  a  sufficient  number  of  tubes  or  flasks  of  agar,  or  serum 
agar,  and  to  incubate  these  as  usual.  With  organisms  that  furnish 
a  prolific  growth,  incubation  for  twenty-four  hours  is  sufficient,  while 
with  the  more  delicate  organisms,  such  as  the  streptococcus  and 
pneumococcus,  it  is  advisable  to  wait  for  forty-eight  to  seventy- 
two  hours.  At  the  expiration  of  this  time  a  small  amount  of  sterile 
saline  solution  (10  c.c.  to  an  ordinary  agar  slant)  is  poured  into  the 
first  tube  and  the  growth  gently  scraped  off  with  a  platinum  loop. 
The  emulsion  is  then  poured  into  the  next,  from  this  into  the  follow- 
ing, and  so  on,  according  to  the  number  of  tubes  or  the  quantity  of 
vaccine  which  is  to  be  prepared. 

1  In  this  connection  I  cannot  condemn  too  strongly  the  use  of  some  of  the 
so-called  polyvalent  and  mixed  vaccines  which  have  been  recently  placed  upon 
the  market  with  the  most  extravagant  claims  for  their  efficacy  in  the  absence 
of  any  proof  of  their  value. 


ACTIVE  IMMUNIZATION  FOR  THERAPEUTIC  PURPOSES     203 

The  general  idea  is  to  make  an  emulsion  at  the  start  that  is  stronger 
than  the  one  desired  in  the  end,  and  subsequently  to  dilute  this  to 
the  required  degree.  The  emulsion  is  transferred  from  the  last  tube  to 
a  sterile  test-tube,  care  being  taken  that  the  fluid  does  not  come  in 
contact  with  the  neck  of  the  tube,  as  otherwise  some  organisms  may 
dry  here  and  subsequently  escape  sterilization.  This  is  then  carried 
out  in  a  water-bath  at  a  temperature  of  from  60°  to  65°  C.  An 
exposure  of  one  hour  is  sufficient,  counting  from  the  time  that 
the  contents  of  the  tube  reach  the  desired  point.  A  number  of 
sterile  glass  beads  are  now  added,  and  the  tube,  tightly  closed  with  a 
sterile  stopper,  shaken  by  hand  or  with  a  machine  for  about  fifteen 
to  twenty  minutes.  The  bacterial  content  is  then  ascertained,  as 
described  above  (see  Preparation  of  Typhoid  Vaccine,  p.  190).  As 
diluent  I  use  an  0.5  per  cent,  solution  of  carbolic  acid,  taking  care 
that  the  final  content  of  the  latter,  in  the  finished  vaccine,  does 
not  fall  below  0.25  per  cent.  The  tube  is  then  allowed  to  stand  on 
end  overnight  (so  as  to  test  the  sterility  of  that  portion  of  the  tube), 
and  a  culture  made  the  next  day,  using  a  good-sized  drop  for  each 
tube,  which  is  conveniently  placed  in  broth  or  milk.  The  preparation 
is  finally  provided  with  a  label,  giving  the  name  of  the  organism, 
and  the  titer  of  the  vaccine  per  1  c.c.  If  desired,  the  vaccine  can, 
of  course,  also  be  put  up  in  glass  beads  or  ampules,  each  containing 
a  single  dose  of  1  c.c.  In  this  form  the  material  is  usually  furnished 
by  the  dealers. 

While  the  common  bacterial  vaccines  may  be  prepared  in  the 
clinical  laboratory  from  autogenous  material,  this  is  out  of  the 
question  in  the  case  of  the  tubercle  bacillus.  Such  a  vaccine  is  best 
obtained  from  the  dealers,  and  is  sold  under  the  name  of  Koch's 
Neu  (new)  Tuberculin,  bacillary  emulsion).  It  is  prepared  by  care- 
fully grinding  fresh  cultures  of  the  bacillus,  after  being  dried  in  the 
vacuum,  in  an  agate  mortar,  or  in  a  specially  constructed  mill,  when 
the  organisms  are  emulsified  in  equal  parts  of  water  and  50  per  cent, 
glycerin  (100  parts  of  each  for  one  part  of  bacilli).  1  c.c.  of  this 
preparation  contains  5  mgrms.  of  bacilli,  and  from  it  the  required 
dilutions  are  made,  care  being  taken  to  sterilize  the  stock  solution 
before  diluting,  by  exposure  to  a  temperature  of  60°  C.  for  one  hour. 
As  diluent  a  0.25  per  cent,  solution  of  lysol  in  physiological  salt 
solution  is  used. 


204  ACTIVE  IMMUNIZATION 

The  Injection. — If  the  vaccine  has  been  put  up  in  bulk  a  small 
quantity  is  poured  into  a  small  medicine  glass  that  has  just  been 
boiled,  and  the  (sterilized)  syringe  charged  from  this;  or  this  is 
filled  from  one  of  the  ampules  directly.  The  skin  is  scrubbed  with 
soap  and  water  and  then  with  alcohol,  or,  as  is  now  recommended, 
painted  with  tincture  of  iodin  at  the  site  of  the  injection.  My 
favorite  site  for  this  is  the  district  over  the  triceps,  into  the  loose 
subcutaneous  tissue.  In  this  region  the  injections  rarely  give  rise 
to  painful  local  reactions,  while  injections  at  a  point  where  the  skin 
is  tightly  bound  down  is  almost  sure  to  cause  a  good  deal  of  avoid- 
able distress.  For  this  reason  also  intradermal  injections  are  to  be 
avoided. 

Dosage  and  Frequency  of  Injection. — As  I  have  already  indicated, 
we  have  as  yet  no  satisfactory  index  to  dosage.  As  the  first  principle 
of  therapeutics  is  noli  nocere,  it  is  advisable  to  begin  with  small 
doses,  i.  e.,  with  quantities  which  past  experience  has  shown  to  do 
no  harm,  so  far  at  least  as  we  can  judge  this  by  clinical  evidence. 

At  the  present  time  the  injections  are  usually  given  about  a  week 
apart,  the  size  of  the  dose  being  increased  at  each  sitting.  If  no 
change  results  from  the  treatment,  larger  doses  may  be  tried;  and  I 
may  say  that  we  have  sufficient  evidence  to  show  that  much  larger 
doses  than  the  maximal  quantities  now  recommended  may  be  given 
in  most  cases.  In  one  or  two  instances  I  have  indeed  received  the 
impression  that  the  patient  owed  his  recovery  from  serious  illness 
to  the  injection  of  a  quantity  of  organisms,  which  was  many  multiples 
of  the  maximal  dose  usually  recommended.  If  the  symptoms  become 
aggravated  the  dose  should  be  diminished  and  the  ascent  carried 
out  less  abruptly  and  possibly  at  somewhat  longer  intervals  (ten  to 
fourteen  days  or  longer) .  Generally  speaking,  in  the  more  acute  cases, 
the  smaller  doses  should  be  chosen,  to  begin  with,  and  the  larger  ones 
reserved  for  the  more  chronic  ones.  There  are,  however,  no  hard- 
and-fast  rules  to  be  laid  down  at  the  present  time.  The  would-be 
immunisator  must  learn  from  experience,  and  should  not  pay  too 
much  attention  to  "negative  and  positive  phases"  when  these  are 
based  on  the  feelings  of  well-being,  and  of  depression  on  the  part 
of  the  patient.  He  should  be  neither  of  too  optimistic  nor  of  too 
pessimistic  a  temperament,  and  should  weigh  the  evidence  with  a 
calm  and  unbiased  mind;  in  other  words,  he  must  be  an  exceptional 
individual.  I  really  know  of  no  field  in  medicine  at  the  present  day 


ACTIVE  IMMUNIZATION  FOR  THERAPEUTIC  PURPOSES     205 

where  it  is  possible  to  draw  so  many  erroneous  conclusions  regarding 
the  value  of  a  therapeutic  agent  as  in  the  domain  of  vaccinotherapy 
in  its  application  to  chronic  infections.  Much  good  can  unquestion- 
ably be  accomplished,  but  we  must  be  careful  not  to  attribute  all 
improvement  to  our  immunizing  efforts. 

Standard  Doses. — As  standard  doses  of  the  different  vaccines,  or 
bacterins,  as  bacterial  vaccines  are  now  termed,  the  following  may  be 
recommended,  bearing  in  mind  what  has  been  said  in  the  foregoing 
lines : 

Staphylococcus  aureus 50,000,000  to     500,000,000  (or  more) 

Staphylococcus  albus  and  citreus     .      .  100,000,000  to  1,000,000,000  (or  more) 

Streptococcus  pyogenes 5,000,000  to     100,000,000  (or  more) 

Gonococcus 5,000,000  to     100,000,000  (or  more) 

Friedlander's  bacillus 10,000,000  to     100,000,000  (or  more) 

Colon  bacillus       .......  10,000,000  to     100,000,000  (or  more) 

Of  the  tubercle  vaccine  it  is  recommended  to  begin  with  very 
small  doses,  i.  e.,  -g^-JinF  *°  TSlhnr  of  a  milligram,  and  to  continue 
the  same  dose  or  gradually  increase  it,  according  to  the  indications 
of  the  individual  case  (see  Tuberculosis). 

Indications  for  the  Use  of  Bacterial  Vaccines. — As  I  have  already 
indicated  in  a  general  way,  bacterial  vaccines  may  be  employed  in 
practically  any  infection  which  has  a  tendency  to  a  certain  degree 
of  chronicity.  It  has  been  recommended  in  the  various  chronic 
infections  of  the  bones  and  joints  (tuberculosis,  osteomyelitis,  gonor- 
rheal  arthritis)  in  the  early  stages  of  pulmonary  tuberculosis,  in 
chronic  gonorrheal  infections,  in  the  colon  bacillus  infections  of  the 
urinary  tract,  in  tubercular  cystitis,  in  the  chronic  Staphylococcus 
infections  of  the  skin  (lupus,  scrofuloderma,  tuberculides),  in  chronic 
inflammation  of  the  middle  ear,  the  antrum,  the  frontal  sinus;  also 
in  endocarditis,  as  a  postoperative  measure  in  connection  with 
empyema,  etc. 

While  acute  infections  have  generally  been  regarded  as  contra-indi- 
cating the  use  of  vaccines,  this  has  largely  been  on  theoretical  grounds. 
Personally,  I  have  gained  the  impression  that  vaccination,  even  in 
such  cases,  could  do  some  good.  However,  it  is  just  in  such  cases 
that  correct  judgment  is  frequently  fallacious  and  difficult. 

To  what  extent  vaccination  may  be  serviceable  as  a  protective 
measure  before  certain  operations  is  difficult  to  say.  Generally 
speaking,  one  should  expect  that  it  might  be  of  use  in  those  cases 


206  ACTIVE  IMMUNIZATION 

in  which  there  is  danger  of  infection,  and  in  which  enough  time  is 
given  before  operations  to  attempt  the  patient's  immunization,  while 
in  urgent  operations  the  measure  would  seem  to  be  contra-indicated 
as  possibly  favoring  infection,  i.  e.,  as  causing  an  immediate,  though 
temporary,  drop  in  the  body's  content  of  protective  substances. 

Results. — It  is  evident  from  what  has  been  said  that  we  know 
too  little  as  yet  of  what  vaccination  can  accomplish  in  the  chronic 
bacterial  infections  to  warrant  any  dogmatic  statements.  The 
amount  of  clinical  material  that  has  been  satisfactorily  studied  is 
entirely  too  small  as  yet,  and  it  seems  to  me  that  we  are  really  not 
entitled  to  say  more  than  that  vaccination  may  do  good  and  should 
be  tried.  But  we  can  neither  state  in  what  percentage  of  cases  it 
will  be  helpful  or  effect  a  cure,  nor  can  we  predict  in  an  individual 
case  what  the  result  will  be.  I  have  seen  excellent  results,  and  no 
results,  in  apparently  similar  cases,  and  I  feel  that  every  unbiased 
observer  has  similar  experiences  to  record.  It  would  after  all  be 
expecting  a  great  deal,  in  the  absence  of  any  more  delicate  indicator 
to  what  is  going  on  in  the  offensive-defensive  interaction  in  the  body, 
than  coarse  clinical  symptoms,  to  be  obliged  to  predict  what  will 
happen  in  a  given  case  and  whether  we  are  doing  the  best  that  can 
be  done. 

While  I  have  pointed  out  in  the  foregoing  pages  that  we  are  not 
yet  in  a  position  where  we  can  speak  definitely  of  the  results  of 
vaccine  treatment  and  its  indications  or  contra-indications,  I  must 
modify  this  statement  somewhat,  so  far  as  tuberculosis  is  concerned, 
and  it  may  not  be  out  of  place  to  consider  this  special  question  by 
itself  and  in  some  detail. 


VACCINE   TREATMENT   OF   TUBERCULOSIS 

The  earliest  attempts  to  influence  the  course  of  tuberculosis  through 
active  immunization  were  made  by  R.  Koch,  and  were  based  upon 
the  observation  that  a  tubercular  guinea-pig  reacts  quite  differ- 
ently to  a  subsequent  inoculation  with  tubercle  bacilli  than  does 
a  normal  animal.  For  whereas  in  the  latter  a  tubercular  ulcer 
develops  at  the  site  of  the  injection  which  does  not  heal,  but  per- 
sists until  the  animal  dies,  local  recovery  occurs  in  the  tubercular 
guinea-pig  without  involvement  of  the  regional  lymph  glands. 


VACCINE  TREATMENT  OF  TUBERCULOSIS  207 

Evidently  then  the  first  inoculation,  even  though  it  leads  to  the 
death  of  the  animal,  produces  a  certain  degree  of  resistance,  which 
the  untreated  animal  does  not  possess,  and  it  very  naturally  occurred 
to  Koch  that  it  might  be  possible  to  utilize  this  observation  as  a  basis 
for  the  treatment  of  human  tuberculosis.  Further  indications  for 
experiments  in  this  direction  were  afforded  by  the  finding,  that, 
whereas  the  injection  of  large  numbers  of  tubercle  bacilli  hastens 
the  death  of  the  tubercular  animal,  small  doses,  frequently  repeated, 
seemingly  have  a  beneficial  influence  both  upon  its  general  condition 
and  upon  the  progress  of  the  lesion  at  the  site  of  the  primary  inocu- 
lation. Since  killed-off  cultures,  however,  though  quite  efficacious, 
were  "apparently  not  resorbed  from  the  point  of  inoculation,  nor 
otherwise  removed,  but  remained  there  undisturbed  and  produced 
abscesses,"  Koch  attempted  so  to  modify  his  vaccine  as  to  separate 
the  curative  from  the  harmful  principle.  The  result  was  his  famous 
tuberculin.  This  was  prepared  by  growing  tubercle  bacilli  for  eight 
weeks  in  4  per  cent,  glycerin-peptone-bouillon,  and  then  concen- 
trating the  culture  at  90°  C.  to  one-tenth  of  its  original  volume.  The 
resultant  material  thus  actually  represents  a  40  per  cent,  glycerin 
extract  of  the  original  culture,  which  is  finally  passed  through  a 
porcelain  filter,  and  is  then  ready  for  use. 

This  is  hardly  the  place  to  narrate  the  history  of  the  introduction 
of  Koch's  tuberculin  into  clinical  use,  the  hopeful  anticipation  with 
which  it  was  received,  and  the  sorrowful  disappointment  that  was 
the  outcome  of  its  earlier  clinical  trials.  Suffice  it  to  recall  that 
the  medical  profession  for  a  while  expected  the  impossible,  and  that 
the  non-realization  of  these  expectations  caused  the  pendulum  to 
swing  the  other  way,  and  to  such  a  degree  in  fact  that  even  now 
the  very  word  "tuberculin"  to  most  minds  suggests  failure.  This 
was  largely  the  outcome  of  the  indiscriminate  use  of  the  substance, 
and  the  fact  that  in  most  cases  the  final  verdict  was  based  upon  a 
trial,  scarcely  extending  over  a  longer  period  than  a  month  or  two 
even  in  cases  where  subsequent  experience  has  shown  that  recovery 
under  its  use  is  possible.  That  this  may  indeed  occur,  and  more 
promptly  so  than  under  an  expectant  plan  of  treatment,  seems  to  be 
undeniable;  but  detailed  clinical  studies  have  shown  that  the  success- 
ful immunization  of  the  tubercular  individual  is  frequently  beset 
with  great  difficulties,  owing  to  the  existence  or  development  of  a 
curious  and  most  remarkable  hypersusceptibility,  in  consequence  of 


208  ACTIVE  IMMUNIZATION 

which  every  injection  is  followed  by  a  reaction  which  is  evidently 
detrimental  to  the  patient. 

In  our  account  of  anaphylaxis  we  have  already  drawn  attention 
to  this  occurrence  and  have  studied  the  mechanism  which  underlies 
its  production.  I  would  merely  emphasize  at  this  place  that  in 
tuberculosis,  probably  more  than  in  any  other  of  the  infectious 
diseases,  anaphylactic  processes  are  responsible  for  many  of  the 
phenomena  which  go  to  make  up  the  clinical  picture  of  the  disease. 
In  the  days  when  Koch's  early  work  on  tuberculin  was  done,  nothing 
was  as  yet  known  of  anaphylaxis,  and  the  symptoms  following  its 
injection  were  generally  attributed  to  associated  toxic  substances. 
Koch  hence  directed  his  efforts  to  the  preparation  of  a  less  toxic 
product,  especially  as  he  had  not  succeeded  in  producing  an 
actual  immunity  in  his  experimental  animals  with  the  old  tuber- 
culin. 

As  it  was  out  of  the  question  to  use  killed-off  cultures  as  such, 
owing  to  the  production  of  abscesses  when  used  hypodermically,  or 
the  formation  of  nodules  in  the  lungs  when  injected  intravenously, 
Koch  resorted  to  the  following  procedure :  Young  cultures  which  had 
been  dried  in  the  vacuum  were  ground  to  pieces  in  a  specially 
constructed  apparatus,  and  the  resultant  powder  shaken  with 
distilled  water,  and  then  centrifugalized.  The  residue  constitutes 
Koch's  so-called  T.  R.  preparation,  while  the  supernatant  fluid  was 
termed  T.  O.  Subsequently  he  brought  out  his  New  (neu)  Tuber- 
culin, which  is  practically  an  aqueous  emulsion  of  the  entire  organ- 
isms, pulverized  to  mere  fragments,  and  preserved  by  the  addition 
of  50  per  cent,  of  glycerin. 

Although  the  use  of  the  old  tuberculin  is  still  continued,  this  new 
product  is  rapidly  gaining  in  favor  and  virtually  corresponds  to  the 
bacterial  vaccines  which  we  have  considered  heretofore.  Its  anti- 
genie  power  is  proved  by  the  fact  that  on  treatment  with  this  material 
the  agglutinin  titer  of  the  patient's  serum  is  frequently  raised  as 
high  as  1 :  500.  This,  to  be  sure,  does  not  constitute  an  index  to  the 
degree  of  immunity  which  is  produced,  but  it  proves  that  the  sub- 
stance in  question  has  the  power  to  bring  about  that  general  allergic 
state  of  which  agglutinin  production  is  one  of  the  possible 
manifestations. 

Dosage  and  Injection. — Old  Tuberculin. — The  old  tuberculin  is  put 
up  in  1  c.c.  and  5  c.c.  ampules.  Unless  a  very  large  number  of 


VACCINE  TREATMENT  OF  TUBERCULOSIS  209 

people  are  to  be  injected  at  one  time  it  is  better  to  use  the  smaller  size. 
From  this  four  dilutions  are  prepared  by  starting  with  a  1  in  10  (A) 
of  the  original  strength,  by  then  making  a  1  in  10  from  this  (B); 
a  1  in  10  from  that  (C),  and  a  1  in  10  from  the  last  (D),  using  sterile 
water  as  diluent,  and  working  with  sterile  glassware.  As  1  c.c.  of 
the  original  product  represents  1000  milligrams  of  the  pure  tuber- 
culin, 1  c.c.  of  dilution  A  will  contain  100  milligrams,  1  c.c.  of  B 
10  milligrams,  1  c.c.  of  C  1  milligram,  and  1  c.c.  of  D  0.1  milligram; 
from  which  latter  further  dilutions  can  be  prepared  according  to  the 
same  plan,  as  desired. 

As  initial  dose,  that  amount  is  suggested  which  a  preliminary 
diagnostic  examination  has  shown  to  produce  a  systemic  reaction 
(see  Tuberculin  Test) .  If  this  is  for  any  reason  omitted,  it  is  best  to 
start  the  patient  with  0.1  milligram,  and  to  increase  the  dose  progres- 
sively at  intervals  which  are  determined  according  to  the  activity 
of  the  reaction,  and  which  accordingly  vary  from  one  to  two  weeks. 
In  the  event  of  a  marked  reaction,  it  is  best  to  repeat  the  last  dose, 
or  to  increase  this  only  very  slightly.  A  reversion  to  a  smaller 
dose  is  to  be  avoided,  and  it  is  better,  if  need  be,  to  wait  a  week 
longer  before  the  next  injection  is  given.  In  the  light  cases  it  is  thus 
possible  to  run  up  to  a  dose  of  1000  milligrams  without  much  trouble, 
while  in  the  presence  of  more  advanced  lesions  this  is  more  difficult; 
when  the  higher  doses  are  reached  the  intervals  between  the  injec- 
tions may  have  to  be  lengthened  to  a  month  or  even  longer.  The 
treatment  is  virtually  considered  at  an  end  when  the  patient 
can  stand  a  dose  of  500  milligrams  without  marked  systemic 
reaction. 

New  Tuberculin. — 1  c.c.  of  new  tuberculin  represents  5  milli- 
grams of  the  dry  powder.  Koch  recommends  that  the  injections 
be  started  with  a  dose  of  0.0025  milligram.  To  this  end  the  original 
product  is  diluted  with  sterile  water  to  the  required  degree,  so  that 
the  amount  to  be  injected  is  less  than  1  c.c.  in  bulk,  as  larger  quan- 
tities favor  the  development  of  local  infiltration.  For  convenience' 
sake  we  can  start  with  a  dilution  of  the  original  product  of  1  in  10 
(A),  1  c.c.  of  which  in  turn  is  diluted  1 :  10  (B),  and  of  this  again  1  c.c. 
in  the  same  proportion  (C) ;  1  c.c.  of  A  then  contains  0.5  milligram, 
1  c.c.  of  B  0.05,  and  1  c.c.  of  C  0.005;  0.5  c.c.  of  the  latter  dilution 
being  thus  the  initial  dose. 

The  injections  are  at  first  given  four  days  a  part,  and  later  when 
14 


210  ACTIVE  IMMUNIZATION 

reactions  begin  to  appear  (i.  e.,  when  amounts  varying  between 
a  tenth  and  one-hundredth  part  of  a  milligram  are  injected)  at 
intervals  of  eight  days,  in  gradually  increasing  amounts,  such  as 
TTO  milligram;  ^nnr>  TOIT>  i~iro>  Tiro*  TTO>  rfcr*  TTO">  TO*  T2o~>  T3o"> 
etc.,  exactly  as  was  customary  with  the  old  tuberculin.  Koch  advo- 
cates that  the  immunization  be  continued  until  the  patients  can 
take  20  milligrams  of  the  dry  powder  without  any  reaction. 

Point  of  Injection. — As  in  the  case  of  the  other  bacterial  vaccines 
the  injections  can  be  conveniently  given  into  the  loose  subcutaneous 
tissue  in  the  district  over  the  triceps,  or  in  the  back  on  a  level  with 
the  angle  of  the  scapula.  Lowenstein  states  that  the  injections 
may  be  advantageously  given  intravenously,  that  the  infiltration 
of  the  skin  is  thus  obviated,  and  that  the  reactions  are  of  briefer 
duration. 

In  advanced  cases  of  pulmonary  tuberculosis  a  combined  treat- 
ment with  old  and  later  with  new  tuberculin  has  been  recommended. 
In  this  connection,  Bandelier  advises  that  when  the  change  from  the 
old  to  the  new  is  made,  to  begin  with  the  two-hundredth  part  of  that 
dose  of  the  old  tuberculin  which  produced  no  reaction. 

While  Koch  emphasizes  the  importance  of  steadily  increasing  the 
dose,  Wright  does  so  only  in  the  beginning;  later  on  he  continues 
with  a  constant  dose.  His  initial  quantity  is  much  smaller  than 
that  recommended  by  Koch,  viz.,  1  c.c.  of  a  dilution  of  the  new 
tuberculin  of  1 :  200000.  Each  dose  is  repeated  a  week  or  ten  days 
apart,  and  then  increased  by  one-fifth  to  one-sixth,  until  1  c.c. 
of  a  dilution  of  1 :  50000  to  1 :  10000  is  reached,  after  which  the 
final  dose  is  continued  (without  further  increase)  for  a  number  of 
months. 

Time  of  Injection. — As  soon  as  reactions  begin  to  appear,  it  is 
advisable  to  give  the  injections  in  the  morning,  so  that  the  patient 
is  not  disturbed  by  the  febrile  movement  during  the  night.  If  this 
is  at  all  marked,  Koch  recommends  that  the  injections  be  stopped, 
and  resumed  two  or  three  days  after  the  temperature  returns  to 
normal,  the  same  dose  being  given  as  the  one  preceding. 

Still  another  procedure  has  been  recommended  by  Wolff-Eisner, 
which  was  planned  with  the  idea  of  developing  receptors  for  the 
tubercular  poison  in  the  cutaneous  connective  tissue,  i.  e.,  in  vitally 
unimportant  structures,  in  which  the  poisons  in  question  that  are 
formed  in  the  diseased  organs  can  be  anchored.  The  method  is  the 


VACCINE  TREATMENT  OF  TUBERCULOSIS  211 

following:  The  existence  of  a  cutaneous  susceptibility  to  the  action 
of  tuberculin  is  first  established  by  intracutaneous  injection  of 
tuberculin  in  different  concentration  (TTOO"  milligram,  or,  if  this 
give  a  negative  result,  of  T^O  or  more).  One-third  of  a  c.c.  of  a 
solution  containing  TTOT  or  TTTO  milligram  to  the  c.c.,  as  the  case 
may  be,  is  then  injected  intracutaneously  at  each  one  of  two  or 
three  different  points,  a  platinum-indium  needle  being  conveniently 
used  for  the  purpose.  These  injections  are  repeated  at  intervals 
of  from  four  to  eight  days,  the  same  dose  being  used  as  long  as  this 
is  followed  by  a  local  reaction.  The  maximal  dose  is  rarely  more 
than  one-tenth  of  a  milligram. 

Indications  and  Centra-indications. — Anyone  who  has  seen  some  of 
the  disastrous  results  which  followed  the  use  of  tuberculin  in  the 
early  days  of  its  history,  will  realize  that  not  all  cases  of  tubercu- 
losis are  suitable  for  the  tuberculin  treatment.  Now  we  know  that 
it  is  best  to  exclude  those  cases  in  which  there  is  any  febrile  move- 
ment of  note,  and  particularly  those  in  which  low  morning  tem- 
peratures alternate  with  correspondingly  high  evening  temperatures; 
then  also  those  in  whom  there  is  evidence  of  active  involvement 
of  the  pleura ;  further,  all  cases  of  pregnancy,  diabetes,  and  epilepsy, 
heart  and  kidney  lesions,  occurring  in  tubercular  subjects,  while  a 
tendency  to  hemorrhage  does  not  in  itself  constitute  a  contra-indica- 
tion.  If,  moreover,  every  injection  is  followed  by  a  marked  reaction, 
and  it  is  impossible  to  obviate  this,  either  by  a  suitable  diminution 
of  the  dose,  or  by  using  one  that  is  larger,  after  giving  the  organism 
time  to  recover  from  the  last  reaction,  it  is  evidently  not  advisable 
to  continue  the  treatment.  Generally  speaking,  Wright's  method, 
or  that  of  Wolff-Eisner,  should  be  employed  in  those  cases  in  which 
we  are  in  doubt  whether  or  not  to  use  tuberculin  at  all.  In  fine, 
I  would  add  that  in  surgical  tuberculosis  the  physician  should  never 
withhold  recognized  surgical  treatment,  hoping  that  immunization 
treatment  alone  will  suffice. 

Reactions. — The  reactions  which  follow  the  use  of  tuberculin  for 
curative  purposes  are  essentially  the  same  as  those  which  are  noted 
when  the  material  is  injected  for  diagnostic  reasons  (which  see). 
There  are,  however,  certain  points  of  difference.  Generally  speaking 
the  reactions  develop  after  a  shorter  time,  which  varies  with  the 
size  of  the  dose.  Following  the  injection  of  3  to  20  milligrams  there 
is  frequently  a  response  as  early  as  eight  hours,  and  after  doses 


212  ACTIVE  IMMUNIZATION 

of  50  milligrams  this  may  even  develop  within  four  or  five  hours. 
The  duration,  moreover,  is  shorter,  so  that  all  the  symptoms  may 
have  disappeared  within  eight  hours,  counting  from  the  time  of 
their  development.  The  response,  both  local  and  systemic,  more- 
over, is  more  intense,  the  former  preceding  the  latter.  As  in  con- 
nection with  the  diagnostic  test,  local  redness  develops  at  the  point 
of  injection  after  one  or  two  hours;  this  is  followed  by  pain  and 
infiltration,  reaching  its  maximum  after  about  twelve  hours  and 
disappearing  only  after  a  number  of  days.  The  systemic  response 
manifests  itself  in  an  initial  chill  or  chilly  sensations,  headache, 
muscle  pain,  and  fever.  The  reaction  reaches  its  height  after  from 
four  to  twelve  or  fourteen  hours  (according  to  the  size  of  the  dose), 
and  then  subsides  so  that  normal  relations  are  restored  within 
twenty-four  or  thirty-six  hours,  the  patient  merely  experiencing  a 
certain  degree  of  lassitude  and  tendency  to  increased  expectoration, 
which  may  continue  for  several  days.  The  height  of  the  temperature 
differs  considerably,  and  while  usually  not  exceeding  102°  F.,  it  may 
reach  103°  and  104°. 

In  especially  susceptible  people,  or  when  the  higher  doses  are 
reached,  the  systemic  symptoms  may  be  much  more  severe  and 
extend  over  many  days,  and  during  such  a  period  it  w^ould,  of  course, 
be  a  mistake  to  repeat  the  injection.  When  these  febrile  periods 
are  very  lengthy  and  accompanied  by  lasting  loss  of  weight  and 
cardiac  disturbance  of  notable  degree,  the  treatment  should  be 
suspended  or  eliminated. 

Results. — So  far  as  the  results  of  the  tuberculin  treatment  go,  so 
much  depends  upon  the  individual  case,  the  duration  of  the  disease, 
the  character  and  seat  of  the  lesion,  the  possibility  of  supplementing 
vaccination  with  adequate  hygienic  treatment,  etc.,  that  from  a 
prognostic  standpoint  every  case  must  be  judged  upon  its  own  merits. 
Suffice  it  to  say  that  the  average  case,  cceteris  paribus,  does  better 
under  immunization  treatment  than  without  it,  and  that  every 
physician  should  recognize  the  rationale  and  value  of  the  method. 
But  I  feel  very  strongly  that  in  order  to  obtain  the  best  results  the 
treatment  should  be  carried  out  either  in  special  institutions  or  by 
men  who  are  thoroughly  familiar  with  the  intricacies  of  immunization 
methods.  As  a  matter  of  fact  the  best  results  have  been  reported 
from  just  such  sources. 

Bandelier  thus  found  that  of  202  cases  of  pulmonary  tuberculosis 


VACCINE  TREATMENT  OF  TUBERCULOSIS  213 

which  had  been  treated  with  tuberculin,  63  per  cent,  no  longer  had 
tubercle  bacilli  in  their  expectoration.  The  best  results,  as  would 
be  expected,  were  obtained  during  the  first  stage  of  the  disease  where 
100  per  cent,  of  the  cases  became  bacilli-free;  among  those  in  the 
second  stage  this  point  was  reached  in  87  per  cent.,  and  among  those 
in  the  third  stage  in  44.2  per  cent.  As  Bandelier  states,  an  equally 
favorable  series  has  not  been  recorded  in  the  literature.  The  patients 
in  question  had  been  treated  in  sanatoria  belonging  to  the  Landes- 
Invalidenversicherung,  of  Berlin.  Koch's  old  tuberculin  had  been 
used  in  all  cases  where  the  physical  examination  suggested  a  tendency 
to  fibrous  changes,  or  in  which,  in  spite  of  extensive  infiltration,  there 
was  little  secretion;  also  in  bone  and  glandular  tuberculosis  and  in 
tubercular  fistulse  of  the  anus.  Otherwise,  i.  e.,  when  there  was 
extensive  softening,  or  when  febrile  reactions  would  have  been 
undesirable,  new  tuberculin  was  employed.  The  outlined  treatment 
with  old  followed  by  new  tuberculin  was  mostly  used  in  advanced 
cases,  and  seems  to  have  furnished  the  best  results,  as  guaged  by  the 
disappearance  of  the  bacilli  in  thirty-eight  of  sixty-nine  cases,  i.  e., 
in  55.07  per  cent.  Considering  the  advanced  character  of  the 
lesion  in  these  individuals,  this  is  indeed  quite  remarkable. 

If  we  contrast  these  findings  with  the  results  of  a  purely  expectant 
(sc.,  hygienic-dietetic)  plan  of  treatment,  where  only  20  per  cent,  of  the 
cases  show  loss  of  bacilli,  no  further  argument  in  favor  of  the  tuber- 
culin treatment  is  required.  It  should  be  remembered,  moreover, 
that  the  actual  results  were  probably  still  better  than  is  suggested 
by  the  above  figures,  if  we  consider  that  the  improvement  continues 
for  three  or  four  months  after  the  treatment  is  suspended.  They 
might  have  been  still  better,  as  Bandelier  suggests,  if  the  limit  of 
immunization,  i.  e.,  the  maximal  dose  of  tuberculin  had  been  higher 
than  10  milligrams,  which  had  been  chosen  as  standard. 

Of  late,  systematic  efforts  have  been  made  to  improve  the  hygienic 
condition  of  the  tubercular  poor,  and  to  give  these  also  the  benefit 
of  the  tuberculin  treatment  when  living  in  their  own  homes.  As  a 
consequence  the  outlook  for  these  unfortunates  has  been  materially 
improved.  Friedrich  thus  records  that  of  700  cases  of  early  tuber- 
culosis which  were  treated  in  this  manner  the  disease  was  arrested 
or  the  patients  much  improved  in  51  per  cent,  of  the  cases.  Similar 
results  have  been  reported  from  other  sources. 


214  ACTIVE  IMMUNIZATION 


ESTIMATION   OF   THE   OPSONIC   CONTENT    OF    THE   BLOOD 
(WRIGHT'S   METHOD) 

Before  concluding  this  chapter  it  may  not  be  out  of  place  to  give 
a  brief  account  of  the  technique  which  Wright  recommended  for 
the  purpose  of  estimating  the  opsonic  index,  but  which  at  present 
has  but  little  more  than  historical  interest,  insofar  as  its  bearings 
on  treatment  or  diagnosis  are  concerned.  The  necessary  apparatus  is 
pictured  in  the  accompanying  illustration  (Plate  III).  It  consists 
of  a  pipette  (a)  for  collecting  corpuscles;  (6)  a  tube  to  receive  the 
blood  to  be  examined,  in  place  in  which  the  blood  capsule  (c)  can 
also  be  used;  and  capillary  pipettes  (d  and  e)  provided  with  rubber 
nipples.  For  purposes  of  incubation  a  special  thermostat  is  recom- 
mended, but  in  its  absence  the  usual  laboratory  incubator  may  be 
employed.  The  actual  "reagents"  are  represented  by  the  patient's 
serum,  a  normal  control  serum,  washed  leukocytes,  and  bacterial 
emulsions. 

Preparation  of  the  Patient's  Serum. — A  small  amount  of  blood 
(about  6  or  8  drops)  is  collected  in  a  little  tube  like  the  one  pictured 
in  Plate  III  at  b,  by  puncturing  the  lobule  of  the  ear  at  its  free 
margin,  in  the  usual  manner,  and  dipping  up  the  blood  as  it  is 
milked  out  by  moderate  pressure.  The  little  tubes  measure  about 
two  inches  in  length  and  have  a  diameter  of  one-quarter  of  an  inch; 
they  may  be  closed  with  a  little  stopper  or  with  adhesive  plaster, 
and  can  then  be  readily  transported.  The  blood  is  allowed  to  clot, 
the  coagulum  separated  from  the  walls  of  the  tube  by  means  of  a 
platinum  wire,  and  the  specimen  centrifugalized  until  the  corpuscles 
have  been  packed  down  and  well  separated  from  the  serum. 

In  place  of  the  tubes  just  described,  which  are  really  most  con- 
venient, Wright  employs  special  capsules  like  the  one  pictured  in 
Plate  III  at  c,  both  ends  of  which  are  sealed.  The  blood  is  collected 
by  puncturing  the  thumb  near  the  root  of  the  nail,  after  having 
previously  allowed  the  arm  to  hang  down  and  then  applying  some 
constriction  behind  the  distal  joint  (tape,  rubber  tubing).  The 
puncture  is  made  with  the  sharp  point  (s)  of  the  straight  limb  of  the 
capsule.  The  sealed  tip  (m)  of  the  bent  limb  is  knicked  off  and  the 
open  end  held  to  the  exuding  drop  of  blood  which  enters  by  capillary 
attraction  until  it  reaches  the  mark  n.  After  knicking  off  the  sealed 


ESTIMATION  OF  THE  OP  SON  1C  CONTENT  OF  THE  BLOOD     215 

tip  at  s,  the  capsule  is  inverted,  when  the  blood  will  occupy  the 
space  above  s.  The  aperture  at  s  is  again  sealed,  and  the  serum 
now  separated  from  the  corpuscles  by  centrifugation,  to  which  end 
the  capsule  is  suspended  on  the  rim  of  the  centrifugalizing  tube  by 
the  bent  limb.  In  the  end  the  tube  is  cut  with  a  file  at  n. 

Preparation  of  the  Normal  Control  Serum. — This  is  collected  in  the 
same  manner  as  the  patient's  serum  and  separated  from  the  corpus- 
cles by  centrifugation.  It  is  best  to  pool  three  or  four  normal  sera, 
viz.,  to  mix  equal  quantities  from  three  or  four  individuals.  If, 
however,  the  serum  of  one  single  person  (the  experimenter,  for 
example)  has  been  thoroughly  studied  and  always  found  normal, 
this  single  serum  may  suffice  for  ordinary  purposes.  Women  during 
menstruation,  hard  workers,  and  individuals  who  are  pale  and  below 
weight,  even  if  otherwise  healthy,  should  not  be  taken  as  controls, 
nor  even  included  in  a  pool.  Occasionally,  apparently  normal 
individuals  are  also  encountered,  who  habitually  have  a  higher 
opsonic  content  than  normal,  and  such  must,  of  course,  also  be 
excluded.  The  process  of  digestion  further  tends  to  increase  the 
opsonic  content  of  the  blood,  so  that  it  is  advisable  to  take  the  blood 
of  the  patient  and  the  pool  approximately  at  the  same  hour  of  the 
day.  As  with  the  patient's  blood  the  control  serum  also  should 
not  be  more  than  twenty-four  hours  old. 

Preparation  of  Washed  Corpuscles  (Leukocytes). — The  blood  is  most 
conveniently  collected  from  the  ear  and  received  in  a  tube  containing 
1.5  per  cent,  sodium  citrate  in  0.9  per  cent,  salt  solution.  The 
amount  will  depend  upon  the  number  of  specimens  that  are  to  be 
prepared;  1  c.c.  is  sufficient  for  at  least  a  dozen  mounts.  Small 
test-tubes  of  5  c.c.  capacity  are  very  convenient.  Clots  must  be 
avoided  and  the  specimen  promptly  discarded  if  the  slightest  coagu- 
lum  forms.  Wright  lets  the  blood  drop  directly  (from  the  finger) 
into  the  citrate  solution,  while  I  use  the  small  tube  a  (Plate  III) 
to  make  the  transfer.  To  prevent  clotting  I  use  a  little  beaker 
with  citrate  saline,  and  between  transfers  always  rinse  the  pipette  in 
this  and  keep  some  of  the  solution  in  the  end,  so  that  the  blood 
immediately  comes  in  contact  with  this;  a  number  of  drops  of 
blood  are  allowed  to  enter  by  capillary  attraction  and  are  then  blown 
out  into  the  little  test-tube;  after  every  addition  the  citrate  tube  is 
closed  with  the  finger  and  inverted  so  as  to  secure  uniform  dilution. 
The  corpuscles  are  then  thrown  down  by  centrifugation,  the  super- 


216  ACTIVE  IMMUNIZATION 

natant  fluid  pipetted  off  and  replaced  with  0.9  per  cent,  saline,  the 
corpuscles  brought  into  suspension  and  again  thrown  down,  when  the 
saline  is  carefully  withdrawn  with  a  capillary  pipette.  Wright  then 
uses  the  superficial  layer  of  corpuscles  only,  as  this  is  especially  rich 
in  leukocytes  (the  leukocytic  cream). 

If  large  quantities  of  leukocytes  are  required,  rabbits  are  injected 
into  the  pleural  cavity  with  5  to  10  c.c.  of  an  emulsion  of  aleuronat 
mush  in  bouillon,  or  0.9  per  cent,  saline,  the  mixture  being  sterilized; 
killed  cultures  (at  120°  C.)  of  staphylococci  (albus  or  aureus)  may 
be  used  for  the  same  purpose.  The  needle  for  injection  should  be 
somewhat  dull,  so  that  bloodvessels  are  not  injured  and  hemor- 
rhage is  prevented.  After  twenty  hours  the  pleural  cavity  will 
contain  an  exudate  rich  in  leukocytes,  which  is  pipetted  off,  placed 
in  citrate-saline,  and  washed  as  described  above. 

Ordinarily  the  leukocytes  should  not  be  kept  longer  than  five  or 
six  hours. 

Preparation  of  Bacterial  Emulsion. — As  the  Wright  technique  neces- 
sitates working  with  uniform  emulsions,  i.  e.,  with  emulsions  in 
which  the  bacteria  ,re  evenly  distributed,  this  step  is  really  the  crux 
of  the  whole  process.  With  certain  organisms,  such  as  the  staphy- 
lococci, the  difficulty  is  not  so  great,  but  with  others,  notably  the 
tubercle  bacillus,  it  is  almost  impossible  to  obtain  uniform  results. 

Staphylococci  and  streptococci  may  be  grown  on  plain  agar,  while 
gonococci,  pneumococci,  and  meningococci  are  cultivated  on  blood 
agar  or  hydrocele  agar.  Small  tubes,  like  the  one  pictured  at  b  (Plate 
III)  are  charged  with  a  little  saline  (0.85  to  1.2  per  cent.).  A  bit 
of  the  culture  is  removed  with  a  platinum  loop  and  gently  rubbed 
against  the  wall  of  the  tube,  at  the  surface  of  the  liquid,  until  a 
uniform  turbidity  results  throughout  the  specimen.  This  is  then 
centrifugalized  for  a  minute  or  two,  so  as  to  remove  clumps  as 
far  as  possible,  and  to  obtain  the  desired  degree  of  density  of  the 
bacterial  emulsion.  This  point  can  only  be  learned  by  experience. 
For  convenience'  sake,  small  glass  capsules  may  be  prepared  con- 
taining emulsions  of  barium  sulphate  of  varying  degrees  of  turbidity, 
and  corresponding  to  bacterial  emulsions  of  standard  strength. 
With  these  the  centrifugalized  specimen  may  be  compared  before 
use  in  the  actual  experiment.  Wright  advocates  an  emulsion  of 
cocci  of  such  strength  that  with  normal  serum  the  average  number 
of  organisms  per  leukocyte  (see  below)  is  about  four  or  five. 


ESTIMATION  OF  THE  OPSONIC  CONTENT  OF  THE  BLOOD     217 

It  has  been  recommended  that  the  cultures  should  not  be  more 
than  twenty-four  hours  old.  This,  however,  is  not  necessary  for 
all  organisms.  Knorr  has  shown  in  my  laboratory  that  the  same 
degree  of  phagocytosis  is  obtained  with  cultures  of  the  staphylococcus 
more  than  a  month  old,  as  with  young  cultures.  In  the  case  of 
the  typhoid  and  the  colon  bacillus,  Wright  recommends  the  use 
of  cultures  only  four  hours  old,  as  with  older  cultures  the  resultant 
spherulation  of  the  organisms  is  such  that  approximative  results 
only  can  be  obtained. 

In  the  case  of  the  tubercle  bacillus,  Cole  obtained  the  best  results  by 
starting  with  living  cultures  on  glycerin  agar,  which  had  been  killed 
by  exposure  to  sunlight  for  twenty-four  hours.  Some  of  the  mate- 
rial is  then  scraped  off,  ground  up  in  an  agar  mortar  with  1.5  per  cent, 
saline  and  centrifugalized  to  remove  clumps.  Cole  states  that  if 
contamination  is  guarded  against  the  supernatant  fluid  may  be  used 
for  at  least  a  month.  I  have  not  had  occasion  to  use  emulsions  pre- 
pared in  this  manner,  and  am  familiar  only  with  emulsions  made 
from  dead  and  ground-up  bacilli.  A  small  quantity  of  this  material 
is  placed  in  an  agate  mortar  and  thoroughly  triturated  with  1.5  per 
cent,  saline,  which  is  slowly  added  drop  by  drop.  The  resultant 
emulsion  may  be  freed  from  coarser  clumps  by  centrifugation,  but 
the  smaller  ones  are  practically  impossible  to  remove.  I  have  worked 
with  heated  and  unheated,  with  extracted  and  non-extracted  bacilli, 
with  0.1  and  1.5  per  cent,  saline,  but  I  have  not  yet  seen  an  emulsion 
of  tubercle  bacilli  that  was  uniform. 

In  the  case  of  the  tubercle  bacillus,  Wright  recommends  that  the 
emulsion  should  be  of  such  strength  that  in  the  actual  experiment 
one  or  two  bacilli  only  are  found  on  an  average  in  each  cell. 

The  Experiment  Proper. — Having  prepared  the  patient's  serum, 
normal  control  serum,  washed  corpuscles,  and  the  bacterial  emulsion, 
these  "reagents"  are  placed  in  a  small  rack,  or  in  a  dishful  of  sand 
covered  with  a  piece  of  white  filter  paper,  perforated  to  receive  the 
tubes,  and  marked  accordingly. 

Mixing  pipettes  (Fig.  d  or  e,  Plate  III)  are  prepared  from  glass 
tubing  having  an  outside  diameter  of  approximately  6  mm.  To 
this  end  pieces  of  tubing  are  cut,  measuring  about  15  cm.  in  length, 
heated  in  the  middle  in  the  flame  of  a  Bunsen  burner  until  soft, 
and  then  drawn  out  after  removal  from  the  flame,  so  that  capillary 
stems  are  obtained  about  10  to  15  cm.  long,  with  a  diameter  of  from 


218  ACTIVE  IMMUNIZATION 

0.5  to  1.0  mm.  The  ends  are  cut  off  square  with  a  fine  file.  The 
tubes  are  marked  about  1  to  2  cm.  from  the  ends  with  a  glass  pencil 
and  before  use  provided  with  medicine-dropper  rubber  nipples.  One 
volume  of  the  leukocytic  "cream"  (see  Preparation  of  Leukocytes, 
above)  is  then  drawn  up  to  the  mark,  followed  by  one  volume  of 
serum  and  one  of  the  bacterial  emulsion,  the  three  portions  being 
separated  from  one  another  by  little  bubbles  of  air  (see  Fig.  d,  Plate 
III).  The  contents  of  the  tube  are  next  blown  out  upon  a  slide 
by  gentle  pressure  upon  the  rubber  nipple,  well  mixed  by  drawing 
them  up  and  down  in  the  capillary  tube,  then  taken  up  in  solid 
column,  and  the  end  sealed  in  the  burner.  The  tubes  are  finally 
incubated  for  fifteen  minutes  at  body  temperature,  which  may  either 
be  done  in  an  ordinary  incubator  or  in  a  special  "opsonifier." 

After  incubation  the  ends  of  the  tubes  are  pinched  off,  drops  are 
mounted  upon  clean  slides,  and  after  having  been  well  mixed  by 
passage  up  and  down  in  the  capillary  pipette,  exactly  in  the  manner 
in  which  the  mixture  was  originally  made,  spreads  on  slides  are 
prepared  by  the  aid  of  the  narrow  edge  of  a  second  slide,  as  in  the 
preparation  of  ordinary  blood  smears.  After  drying  in  the  air  the 
specimens  may  be  stained  with  aqueous  methylene  blue,  with  some 
polychrome  dye,  such  as  Jenner's,  Hastings',  Wilson's,  or  Giemsa's 
stain,  or  with  Borrell's  carbol-thionin,1  the  specimens  being  fixed 
with  absolute  methyl  alcohol,  if  aqueous  stains  are  to  be  employed, 
while  this  is,  of  course,  unnecessary  in  the  case  of  alcoholic  mixtures. 
Tubercle  specimens  are  fixed  by  immersion  for  one  minute  in  a 
saturated  aqueous  solution  of  mercuric  chloride.  They  are  then 
washed  off  in  water,  stained  with  steaming  carbol  fuchsin,  washed 
with  water,  decolorized  in  2.5  per  cent,  sulphuric  acid,  treated  with 
4  per  cent,  acetic  acid  solution  to  destroy  the  red  cells,  again  washed 
in  water,  counterstained  with  1  per  cent,  aqueous  methylene  blue, 
washed  once  more,  and  then  allowed  to  dry. 

The  average  number  of  bacteria  per  leukocyte  (phagocytic  index) 
is  finally  ascertained  by  going  over  at  least  a  hundred  cells,  and  the 
opsonic  index  then  calculated  by  dividing  the  patient's  phagocytic 
index  by  the  normal,  which  is  taken  as  unity.  Example :  Supposing 

1  A  saturated  solution  of  thionin  in  distilled  water  is  precipitated  with  a  10 
per  cent,  soda  solution;  the  precipitate  is  collected  on  a  small  filter,  washed  twice 
with  distilled  water,  and  then  dissolved  in  5  per  cent,  carbolic  acid  solution 
(1  gram  :  100  c.c.).  The  solution  must  always  be  filtered  before  use. 


ESTIMATION  OF  THE  OP  SONIC  CONTENT  OF  THE  BLOOD     219 

that  with  the  patient's  serum  the  average  number  of  organisms  per 
cell  was  5  and  with  the  normal  serum  10;  then  from  the  equation 
10  :  1  :  :  5  :  x,  it  would  follow  that  the  opsonic  index  is  0.5. 

When  Wright's  studies  on  the  opsonins  first  appeared  they 
attracted  a  great  amount  of  attention.  This  was  largely  owing  to  the 
fact  that  the  author  attached  a  significance  to  his  observations 
which,  if  justified,  would  have  meant  an  enormous  advance  not 
only  in  the  diagnosis  of  certain  bacterial  infections,  but  also  in  their 
treatment.  I  cite  some  of  his  more  important  diagnostic  deductions : 

1.  Conclusions   which    can    be    arrived    at    when    we    have   at 
disposal  the  results  of  a  series  of  measurements  (opsonic  determi- 
nations) : 

(a)  When  a  series  of  measurements  of  the  opsonic  power  of  the 
blood  reveals  a  persistingly  low  opsonic  power  with  respect  to  the 
tubercle  bacillus,  it  may  be  inferred,  in  the  cases  in  which  there  is 
evidence  of  a  localized  bacterial  infection  which  suggests  tuber- 
culosis, that  the  infection  in  question  is  tuberculous  in  character. 

(6)  When  repeated  examination  reveals  a  persistently  normal 
opsonic  power  with  respect  to  the  tubercle  bacillus,  the  diagnosis 
of  tubercles  may  with  probability  be  excluded. 

(c)  When  there  is  revealed  by  a  series  of  blood  examinations  a 
constantly  fluctuating  opsonic  index  the  presence  of  active  tuber- 
culosis may  be  inferred. 

2.  Conclusions  which  may  be  derived  at  where  we  have  at  disposal 
the  result  of  an  isolated  blood  examination: 

(a)  When  an  isolated  blood  examination  reveals  that  the  tuber- 
culo-opsonic  power  of  the  blood  is  low,  we  may — according  as  we 
have  evidence  of  a  localized  bacterial  infection  or  of  constitutional 
disturbance — infer  with  probability  that  we  are  dealing  with  tuber- 
culosis— in  the  former  case  with  a  localized  tuberculous  infection, 
and  in  the  latter  with  an  active  systemic  infection. 

(6)  When  an  isolated  blood  examination  reveals  that  the  tuberculo- 
opsonic  power  of  the  blood  is  high,  we  may  infer  that  we  have  to 
deal  with  a  systemic  tuberculous  infection  which  is  active,  or  has 
recently  been  active. 

(c)  When  the  tuberculo-opsbnic  power  is  found  normal  or  nearly 
normal,  while  there  are  symptoms  which  suggest  tuberculosis,  we 
are  not  warranted,  apart  from  the  further  test  described  below,  in 
arriving  at  a  positive  or  a  negative  diagnosis. 


220  ACTIVE  IMMUNIZATION 

The  further  criterion  to  which  reference  has  been  made  in  the 
preceding  paragraph  is  the  following: 

When  a  serum  is  found  to  retain  in  any  considerable  measure, 
after  it  has  been  heated  to  60°  C.  for  ten  minutes,  its  power  of  incit- 
ing phagocytosis,  we  may  conclude  that  "incitor  elements"  (immune 
opsonins)  have  been  elaborated  in  the  organism  either  in  response 
to  auto-inoculations,  occurring  spontaneously  in  the  course  of  tuber- 
culous infection,  or,  as  the  case  may  be,  under  the  artificial  stimulus 
supplied  by  the  inoculation  of  tubercle  vaccine. 

The  above  considerations  apply  also  in  the  case  of  other  bacterial 
infections,  and  in  the  examination  of  exudates  as  well. 

As  Wright  regarded  the  opsonic  index  as  an  indicator  of  the 
degree  of  immunity  which  develops  as  the  result  of  bacterial  vacci- 
nation (which  see),  he  advocated  that  the  dosage  and  frequency  of 
injection  should  be  controlled  by  opsonic  determinations.  Accord- 
ing to  his  teachings  the  injection  of  a  dose  of  vaccine  is  followed 
by  a  decrease  of  the  opsonins  (negative  phase),  which  is  of  variable 
degree  and  duration,  according  to  the  amount  injected.  This  is 
followed  by  an  increase  (positive  phase)  coincidently  with  which 
there  is  a  corresponding  improvement  in  the  patient's  condition. 
The  idea  of  proper  vaccination,  then,  is  to  so  gauge  and  interspace 
the  different  doses  that  a  negative  phase  is  obviated  as  far  as  possible 
and  a  "high  tide"  of  increased  opsonic  content  secured. 

It  would  lead  too  far  to  discuss  the  teachings  of  Wright  in  any  detail 
at  this  place;  suffice  it  to  say  that  nearly  all  investigators  who  have 
busied  themselves  with  his  technique  have  come  to  the  conclusion 
that  the  unavoidable  sources  of  error  are  such  that  accurate  results 
cannot  be  obtained.  As  a  consequence  its  application  looses  much 
of  its  raison  d'etre,  and  at  the  present  time  there  are  few  outside  of 
Wright's  own  circle  who  are  influenced  in  either  diagnosis  or  treat- 
ment by  the  opsonic  index.  But  this  failure  does  not  in  the  least 
diminish  the  importance  of  the  principle  of  bacterial  vaccination, 
a  principle  which  had,  however,  been  firmly  established  long  before 
the  opsonins  were  discovered. 

It  would,  of  course,  be  most  desirable  to  possess  an  index  to 
dosage  and  frequency  of  injection  in  vaccination,  but  a  consideration 
of  what  has  already  been  said  regarding  the  aggressive  forces  of 
the  bacteria  will  at  once  suggest  that  even  if  it  could  be  possible 
to  estimate  the  "opsonic  index"  with  accuracy,  this  alone  would 


ESTIMATION  OF  THE  OP  SON  1C  CONTENT  OF  THE  BLOOD     221 

scarcely  be  of  much  value  in  the  treatment  of  infections.  For 
unless  we  can  influence  the  aggressive  forces  of  the  invading  organ- 
isms and  notably  their  capsule-forming  power,  the  production  of  a 
high  content  of  opsonins  in  itself  would  lead  to  nothing. 

In  conclusion,  I  would  briefly  call  attention  to  the  fact  that  in 
the  early  days  of  the  opsonic  "high  tide"  I  advocated  a  different 
method  of  estimating  the  opsonins,  which  was  based  upon  the  prin- 
ciple of  dilution,  and  I  note  with  satisfaction  that  this  principle  is 
now  utilized  in  practically  all  laboratories  (outside  of  Wright's)  in 
which  opsonic  studies  are  being  carried  on. 


CHAPTER  XIII 
PASSIVE  IMMUNIZATION 

WHILE  active  immunization  is  the  procedure  par  excellence  to  be 
employed  for  prophylactic  purposes,  or  in  the  treatment  of  those 
infections  which  are  characterized  by  a  chronic  course,  the  indi- 
cations for  passive  immunizations  are  essentially  afforded  by  the 
acute  infections,  the  idea  being  that  in  these  the  necessary  time  may 
not  be  available  for  the  formation  of  protective  antibodies,  or  that 
these  are  not  furnished  in  sufficient  quantity  by  the  infected  organism 
itself.  The  plan,  then,  is  to  introduce  these  principles  from  without, 
either  in  the  form  of  antitoxic  sera,  or  of  bacteriolytic-bacteriotropic 
sera,  as  the  case  may  be.  That  such  sera  may  at  times  be  serviceable 
also  for  prophylactic  purposes,  goes  without  saying,  but  it  is  natural 
that  their  value  from  this  standpoint  should  be  limited.  For  whereas 
in  the  actively  immunized  organism  the  entire  defensive  mechanism 
is  thrown  into  action,  and  remains  in  action,  often  for  a  considerable 
length  of  time,  the  protective  principles  which  we  introduce  from 
without  are  after  all  limited  in  quantity,  and  are,  no  doubt,  elimi- 
nated or  destroyed  after  a  relatively  short  time.  If  such  sera  are 
administered  at  a  time,  however,  when  the  organism  has  just  become 
infected,  or  is  immediately  threatened  with  infection,  their  use  is 
unquestionably  rational,  and  frequently  of  great  value. 

As  regards  the  mode  of  action  of  the  two  types  of  sera,  which  are 
available  for  passive  immunization,  I  would  merely  recall  that  those 
organisms  which  are  strong  toxin  producers  are  also  of  a  low  grade 
of  infectiousness,  and  that  the  macroorganism  can  usually  overcome 
the  infection  as  such  without  much  difficulty,  if  it  is  protected 
against  the  harmful  effect  of  the  toxins.  In  tetanus  the  infection 
is  indeed  usually  already  under  control  before  the  toxins,  which 
have  been  liberated,  can  exercise  their  fatal  action.  With  the  true 
parasites  and  semiparasites,  on  the  other  hand,  where  toxins  either 
play  no  role  or  only  a  limited  role,  the  bacteriolytic  sera  would, 
a  priori,  be  expected  to  be  of  service,  but,  unfortunately,  their  actual 


DIPHTHERIA  223 

therapeutic  value  is  very  small.  In  our  discussion  of  the  different 
sera,  we  shall  accordingly  only  afford  a  limited  space  to  the  latter, 
and  largely  confine  our  attention  to  those  possessing  marked  anti- 
toxic properties,  the  discovery  of  which  ranks  as  one  of  the  most 
important  in  the  science  of  medicine.  The  sera  which  here  enter 
into  consideration  will  be  discussed  under  the  heading  of  the  diseases 
against  which  they  are  directed. 


ANTITOXIC    IMMUNIZATION 

DIPHTHERIA 

After  Roux  and  Yersin  had  shown  that  the  clinical  picture  of 
diphtheria  is  due  to  the  action  of  a  soluble  toxin  which  is  secreted 
by  the  corresponding  organisms  (1888),  v.  Behring  found  that 
animals  which  have  been  immunized  against  diphtheria  are  thus 
rendered  resistant  to  the  toxin  in  question,  and  that  the  blood  of 
such  animals  contains  a  principle  which  can  be  transferred  to  other 
animals  and  can  protect  these  against  subsequent  infection,  or  cure 
this,  as  the  case  may  be  (1890).  This  principle  he  termed  antitoxin. 

The  first  attempt  to  apply  this  important  discovery  to  the  cure  of 
diphtheria  in  the  human  being  was  made  in  Berlin  in  v.  Bergmann's 
clinic  (1891).  The  results,  while  suggestive,  were  not  altogether 
satisfactory,  however,  as  the  serum  which  was  then  available  was 
too  weak  and  the  dosage  too  small.  But  subsequent  investigations 
by  a  number  of  different  observers,  notably  Roux,  Ehrlich,  Kossel 
and  Wassermann,  Aronson  and  Baginsky,  etc.,  supported  v.  Behring's 
claims  in  their  entirety  and  demonstrated  conclusively  that  one  of 
the  most  fearful  and  most  intractable  diseases  to  which  the  human 
being  is  subject  had  indeed  been  conquered.  Since  then  diphtheria 
antitoxin  has  been  the  means  of  saving  untold  thousands  of  lives 
which  otherwise  would  have  been  doomed,  and  has  thus  proved 
one  of  the  greatest  blessings  to  the  entire  civilized  world. 

While  people  still  die  of  diphtheria  at  the  present  day,  this  is 
largely  owing  to  ignorance  or  indifference  on  the  part  of  those  to 
whom  the  medical  profession  must  after  all  look  for  the  earliest 
diagnosis  of  the  disease,  or  at  least  for  the  recognition  of  those 
symptoms  which  should  serve  as  danger  signals,  i.  e.,  the  parents 


224  PASSIVE  IMMUNIZATION 

and  guardians  of  young  children,  and  of  those  who  are  so  situated 
that  they  cannot  look  after  themselves.  Of  physicians,  we  may  hope, 
there  are  none  who  at  the  present  day  would  withhold  from  their 
patients  what  the  scientific  world  has  come  to  recognize  as  the  most 
potent  and  important  curative  agent  in  the  management  of  the 
disease  in  question.  If,  by  any  chance,  however,  there  should  be 
such  a  person,  then  the  laity  should  realize  that  the  non-use  of 
diphtheria  antitoxin,  in  the  absence  of  special  indications  to  the 
contrary,  constitutes  sufficient  evidence  of  inefficiency  on  the  part 
of  the  practitioner  to  warrant  his  prosecution  in  the  courts. 

Preparation  of  Diphtheria  Antitoxin. — While  the  earliest  attempts 
at  immunization  were  made  with  the  serum  of  some  of  the  smaller 
laboratory  animals  (sheep  and  goats),  it  soon  became  apparent  that 
from  such  sources  a  sufficient  supply  could  not  conveniently  be 
secured,  and  at  the  present  time  the  horse  is  universally  employed 
as  antitoxin  producer. 

The  animals  which  are  chosen  for  this  purpose  are  first  tested 
with  tuberculin  and  mallein  for  freedom  from  tuberculosis  and 
glanders,  and,  further,  receive  an  injection  of  tetanus  antitoxin  to 
counteract  any  accidental  infection  of  this  kind  which  might  acci- 
dentally occur  during  the  period  of  time  that  the  animals  are  fur- 
nishing antitoxin.  They  are  well  fed  and  groomed,  and  every 
effort  in  short  made  to  maintain  them  in  the  best  condition  possible. 

For  purposes  of  immunization  the  toxin  furnished  by  a  special 
strain  of  the  diphtheria  bacillus  is  now  used  the  world  over.  This 
strain  has  been  studied  with  special  care  by  Park  and  Williams 
(New  York),  whose  names  it  bears,  and  is  grown  in  2  per  cent, 
peptone  nutrient  bouillon  of  an  alkalinity  corresponding  to  8  c.c. 
of  normal  soda  solution  per  liter  (above  the  neutral  point  to  litmus), 
the  broth  being  beef-broth,  and  the  peptone  the  usual  preparation 
of  Witte.  This  medium  is  placed  in  comparatively  thin  layers  in 
wide-mouthed  Erlenmeyer  flasks,  and  kept  at  a  temperature  of 
from  35°  to  36°  C.  At  the  end  of  a  week  the  toxin  production  has 
reached  its  maximal  point,  when  the  cultures  are  tested  in  reference 
to  their  purity,  and  are  killed  off  by  the  addition  of  10  per  cent,  of 
a  5  per  cent,  solution  of  carbolic  acid.  After  standing  for  forty- 
eight  hours,  most  of  the  bacilli  have  settled  to  the  bottom,  the  clear 
supernatant  fluid  is  filtered  through  sterile  filter  paper  and  is  stored 
in  full  bottles  in  the  refrigerator.  Before  use  it  is  tested  on  guinea- 


DIPHTHERIA  225 

pigs.     If  the  material  contains  an  adequate  amount  of  toxin,  less 
than  0.01  c.c.  should  kill  an  animal,  weighing  about  250  grams. 

Since  the  injection  of  the  crude  toxin  often  gives  rise  to  quite 
severe  local  as  well  as  systemic  reactions,  various  attempts  have 
been  made  to  so  modify  the  material  as  to  diminish  this  feature  as 
far  as  possible  without  interfering  with  its  antigenic  value.  This 
has  been  accomplished,  in  a  measure,  by  giving  the  horse  a  large 
dose  of  antitoxin  mixed  with  the  first  three  doses  of  toxin.  In  the 
laboratories  of  the  Health  Department  of  New  York  City  the 
animals  thus  receive  as  initial  dose  an  amount  of  toxin  sufficient  to 
kill  5000  guinea-pigs  (average  weight  250  grams),  i.  e.,  about  20 
c.c.,  and  mixed  with  this  10,000  units  of  antitoxin.  This  injection 
(given  subcutaneously)  is  followed  by  a  febrile  reaction  which  lasts 
for  three  to  five  days,  when  a  second  injection  of  a  slightly  larger 
dose  is  given,  and  after  a  similar  period  of  time  a  third  one,  both 
being  accompanied  by  a  dose  of  10,000  antitoxin  units,  as  in  the  first 
instance.  After  that  the  immunization  is  continued  with  increasing 
doses  of  toxin,  given  by  itself,  and  at  intervals  of  five  to  eight  days, 
until  at  the  end  of  two  months  from  ten  to  twenty  times  the  original 
amount  is  given  (Park).  If  during  this  period  the  animal  should 
at  any  time  react  unduly  by  fever,  or  if  any  infiltration  should  occur, 
it  is  recommended  to  resume  the  combined  administration  of  toxin 
with  antitoxin,  as  in  the  beginning.  At  the  expiration  of  six  weeks 
or  two  months  the  animal's  blood  is  tested  for  its  content  in  anti- 
toxin. If  by  that  time  this  has  reached  a  titer  of  100  to  150  units 
the  animal  may  be  expected  to  ultimately  furnish  a  serum  of  moderate 
strength.  If  high-grade  sera  only  are  desired,  it  is  needless  to  con-  I  f 
tinue  with  any  animal  that  at  this  period  does  not  give  a  titer  which  \ 
is  higher  than  150. 

After  this  test-bleeding,  immunization  is  further  continued  with 
increasing  doses,  at  intervals  of  three  days  to  a  week,  until  the 
animal  furnishes  a  serum  with  the  titer  that  is  desired,  or  until  this 
can  no  longer  be  increased.  At  the  end  of  three  months  two  or 
three  animals  out  of  fifteen  or  twenty  will  give  a  titer  of  about  500 
units,  and  half  of  the  total  number  one  of  180  to  200.  Further 
injections  may  increase  the  production  still  further,  but  it  is  note- 
worthy that  values  of  500  to  600  units  are  rare.  Higher  values  than 
1000  are  very  uncommon,  and  Park  states  that  of  his  horses  not  a 
single  one  ever  yielded  2000  units.  In  those  animals,  moreover, 
15 


226  PASSIVE  IMMUNIZATION 

which  do  yield  exceptionally  high  values,  the  high  tide  of  antitoxin 
production  is  only  of  brief  duration.  As  the  maximum  production  of 
antitoxin  even  under  the  most  favorable  conditions  does  not  con- 
tinue beyond  a  few  months,  and  is  then  followed  by  a  decline,  in 
spite  of  further  immunization,  it  is  advisable  to  give  the  animals 
a  period  of  three  months'  rest  in  every  twelve  that  they  are  in  service. 
If  this  is  done,  the  best  horses  furnish  high-grade  serum  during  their 
periods  of  treatment  for  from  two  to  four  years  (Park).  As  6000  c.c. 
of  blood  may  be  taken  from  an  animal  at  intervals  of  one  month, 
it  will  be  seen  that  the  yield  per  year  amounts  to  from  36  to  54 
liters,  allowing  for  the  three  months'  trial  immunization  during  the 
first  year  and  three  months  of  rest. 

When  it  is  desired  to  draw  off  some  of  the  blood  a  superficial 
vein  of  the  neck  is  punctured  with  a  fair-sized,  sharp-pointed  cannula 
and  the  blood  allowed  to  flow  through  an  attached  tube  into  large 
Erlenmeyer  flasks,  special  pains  being  taken  to  work  aseptically 
throughout  the  whole  procedure.  The  flasks  are  placed  in  a  slanting 
position  before  the  blood-clots,  and  kept  in  a  cool  room  for  three  or 
four  days,  when  the  serum  which  has  separated  out  is  pipetted  off 
and  stored,  preliminary  to  a  bacteriological  examination  and  the 
determination  of  its  titer,  after  which  it  is  filled  into  little  ampoules, 
or  into  individual  syringes,  as  the  case  may  be,  and  is  then  ready  for 
use.  In  Germany,  carbolic  acid  is  used  as  a  preservative,  to  the 
extent  of  0.5  per  cent.,  while  in  the  United  States,  0.4  per  cent, 
tricresol  is  preferred,  unless  indeed  the  serum  is  used  as  such,  which 
is  now  frequently  the  case.  The  individual  package  is  appropriately 
labelled,  and  the  date  indicated,  after  which  it  should  no  longer  be 
used.  This  is  necessary,  as  the  antitoxic  titer  diminishes  in  the 
course  of  time.  Park  states  that  the  serum  which  is  prepared  by 
the  New  York  Board  of  Health  remains  within  10  per  cent,  of  its 
original  strength  for  at  least  two  months,  when  kept  from  the  access 
of  air  and  light  in  a  cool  place,  but  that  within  a  year  the  loss  in 
strength  may  amount  to  40  per  cent. 

Determination  of  the  Titer. — In  the  study  of  the  titer  of  diphtheria 
antitoxin  the  following  standards  are  employed:  As  unit  of  diph- 
theria toxin  we  designate  that  quantity  expressed  in  fractions  of  a 
c.c.,  which  is  just  sufficient  to  kill  a  guinea-pig  weighing  250  grams 
in  the  course  of  four  or  five  days.  In  other  words,  the  single  lethal 
dose  constitutes  the  unit. 


DIPHTHERIA  227 

A  toxin  broth  which  contains  100  units  per  c.c.,  v.  Behring  has 
termed  a  normal  toxin  solution,  and  he  designates  this  by  the  formula 
DTN,  M25o,  which  signifies:  diphtheria  toxin,  single  normal,  in 
reference  to  a  guinea-pig  weighing  250  grams.  Of  this  1  c.c.  would 
suffice  to  kill  one  hundred  guinea-pigs  and  0.01  c.c.  a  single  animal. 
A  double  normal  toxin  solution,  DTN2M25o,  would  accordingly 
be  one  of  which  one-half  that  dose,  i.  e.,  0.005  c.c.,  would  suffice  to 
kill  a  guinea-pig  of  standard  weight.  A  unit  of  antitoxin,  on  the 
other  hand,  is  that  quantity  which  is  capable  of  neutralizing  100 
units  of  toxin,  and  we  designate  as  normal  serum  one  of  which 
1  c.c.  will  neutralize  1  c.c.  of  normal  toxin  solution,  i.  e.,  100  units 
of  toxin. 

1  c.c.  normal  antitoxin  serum  =  1  c.c.  normal  toxin  solution  is 
sufficient  to  protect  100  guinea-pigs,  each  against  a  single  lethal  dose 
—0.01  c.c. — of  normal  toxin). 

Ehrlich  designates  as  Lf  (limes  =  limit)  that .  quantity  of  toxin 
which  when  mixed  with  one  unit  of  antitoxin  and  injected  subcu- 
taneously  into  a  guinea-pig  weighing  250  grams  will  kill  the  animal 
in  four  or  five  days,  while  he  denotes  the  quantity  which  is  just 
neutralized  by  1  unit  of  antitoxin,  and  which  will  hence  not  kill 
the  animal  when  injected  together  with  the  toxin  as  L0. 

To  determine  the  strength  of  a  given  serum  it  is  for  practical 
purposes  only  necessary  to  inject  a  series  of  guinea-pigs  subcuta- 
neously,  each  with  a  mixture  containing  say  100  units  of  toxin  and 
varying  quantities  of  the  serum  under  consideration.  If  the  animal 
dies  within  the  first  days  the  amount  of  antitoxin  was  evidently 
not  sufficient  to  neutralize  all  the  toxin,  and  the  serum  hence  had  a 
titer  lower  than  1  unit  to  the  c.c.  If  death  takes  place  on  the  fifth 
or  sixth  day  the  antitoxin  content  is  just  a  unit,  and  if  the  animal 
does  not  die  at  all,  it  must  have  been  stronger  than  this.  Supposing 
that  1  c.c.  of  the  serum  in  a  dilution  of  1 : 1000  had  been  sufficient 
to  delay  death  until  the  fifth  or  sixth  day,  then  0.001  c.c.  of  the 
concentrated  serum  would  represent  one  antitoxin  unit,  and  its 
actual  titer  would  hence  be  1000  units  per  c.c. 

In  Germany  the  production  of  antitoxin  is  carefully  supervised 
by  the  government  and  every  preparation  tested  in  the  Institute 
for  Experimental  Therapy,  of  which  Ehrlich  is  the  head.  In  the 
United  States  there  are  now  also  stringent  laws  regulating  its  prepar- 
ation, and  specimens  are  purchased  from  time  to  time  in  the  open 


228  PASSIVE  IMMUNIZATION 

market  for  examination  at  the  hygienic  laboratories  of  the  Public 
Health  and  Marine  Hospital  Service. 

In  the  United  States  diphtheria  antitoxin  is  now  marketed  in 
500,  1000,  2000,  3000,  4000,  5000,  and  10,000  unit  doses. 

The  Injection. — The  injections  are  usually  given  into  the  loose 
subcutaneous  tissue  between  the  shoulder-blades,  into  the  abdominal 
walls,  or  into  the  district  overlying  the  triceps.  The  skin  should  be 
scrubbed  with  soap  and  water  and  then  with  alcohol,  or  as  is  now 
also  advised,  merely  painted  with  tincture  of  iodin  about  the  point 
of  injection.  If  a  separate  syringe  be  used  this  should,  of  course,  be 
sterilized  by  boiling,  but  in  the  United  States  the  manufacturers 
now  send  the  antitoxin  out  in  separate  syringes  which  are  already 
sterile  and  ready  for  immediate  use. 

Of  late  it  has  been  suggested  that  a  more  powerful  effect  may  be 
secured  if  the  antitoxin  is  administered  intramuscularly,  or,  still 
better,  intravenously.  To  this  there  can  be  no  objection  if  the  amount 
of  preservative  that  is  thus  injected  at  one  time  remains  within  the 
limits  of  the  permissible  dose.  In  Heubner's  clinic  18  c.c.  of  serum 
containing  0.5  per  cent,  carbolic  acid  have  thus  been  injected  at 
one  time  and  the  dose  repeated  within  twenty-four  hours.  With 
us,  in  the  United  States,  where  no  preservative  is  frequently  used, 
even  this  objection  does  not  exist.  The  advantage  of  the  intravenous 
over  the  subcutaneous  method  of  administration  has  been  clearly 
shown  by  Berghans,  who  found  in  the  animal  experiment  that  whereas 
40  units  of  antitoxin  were  necessary  to  prevent  the  death  of  a  guinea- 
pig  when  given  subcutaneously,  0.08  was  sufficient  when  injected 
directly  into  the  circulation,  the  amount  of  toxin  having  been  the 
same  in  both  instances.  The  importance  of  resorting  to  this  method 
of  administration  is  further  emphasized  by  the  observation  made 
in  the  Danish  Serological  Institute  that  following  the  subcutaneous 
use  of  the  antitoxin  this  does  not  reach  its  maximum  in  the  circu- 
lation until  the  second  or  third  day.  Eckert  thus  very  properly 
insists  that  the  intravenous  method  is  the  method  par  excellence 
to  be  employed,  and  that  with  its  general  adoption  the  death-rate 
from  diphtheria  will  be  lowered  still  farther  (see  below). 

Dosage  and  Uses. — In  the  treatment  of  diphtheria  by  antitoxin 
it  is  important  to  bear  in  mind  that  the  quantity  of  toxin  that  is 
produced  and  likely  to  be  absorbed  is,  c&teris  paribus,  the  greater 
the  longer  the  duration  of  the  disease,  and  that  the  union  of  the 


DIPHTHERIA  229 

toxin  with  the  receptors  of  sensitive  cells  will  be  the  firmer  the 
longer  this  has  lasted.  It  follows  that  large  doses  will  be  required, 
if  the  patient  first  comes  under  observation  after  the  disease  has 
already  existed  for  a  number  of  days,  and  that  in  the  presence  of 
toxic  symptoms,  indicating  that  toxin  has  already  been  anchored 
by  sensitive  cells,  very  large  doses  only  can  be  expected  to  be  helpful. 
It  is  accordingly  recommended  that  the  physician  should  not  delay 
the  use  of  antitoxin  until  a  bacteriological  examination  has  been 
made,  but  to  resort  to  it  whenever  diphtheria  is  suspected.  This 
rule  is  indeed  the  only  natural  one  to  follow. 

As  to  the  size  of  the  initial  dose,  the  last  word  has  probably  not 
yet  been  spoken.  In  the  earlier  days  of  antitoxin  treatment  100  to 
200  units  were  recommended,  but  since  then  there  has  been  a  ten- 
dency to  ever-increasing  amounts,  and  in  the  United  States  3000 
units  may  now  be  regarded  as  an  average  dose  in  cases  of  moderate 
severity.  If  a  longer  interval  than  twenty-four  hours  has  elapsed 
before  the  patient  is  first  seen,  the  dose  should  be  still  larger,  and  if 
threatening  symptoms  of  any  kind  exist  the  physician  should  not 
hesitate  to  inject  10,000  units  or  more  at  the  time  of  his  first  visit. 
Some  writers,  indeed,  have  used  much  larger  amounts  (up  to  50,000 
to  100,000  units)  and  have  reported  favorable  results  in  the  most 
desperate  cases. 

Following  the  first  injection  the  antitoxin  is  continued  at  intervals 
of  twelve  to  twenty-four  hours,  until  the  disease  is  evidently  under 
control,  and  I  would  emphasize  once  more  that  much  time  may  be 
saved  if  the  injections  are  given  intravenously,  or  even  intramus- 
cularly. In  severe  cases  the  subcutaneous  administration  should 
unquestionably  be  abandoned,  since  the  absorption  owing  to  the 
lowered  blood  pressure  must  then  be  still  slower  than  in  a  healthy 
individual,  where  the  maximal  blood  content  in  antitoxin  is  scarcely 
reached  before  the  third  day. 

Total  Quantity  that  may  be  Administered. — As  to  the  quantity 
of  antitoxin  which  may  be  administered  in  the  course  of  the  malady 
there  is  apparently  no  limit.  Bankier  thus  reports  the  case  of  a 
child  in  which  72,000  units  were  given,  and  in  which  recovery  oc- 
curred in  spite  of  the  most  ominous  symptoms  (profuse  nose-bleed, 
extensive  hemorrhagic  ecchymoses  of  the  skin,  paresis  of  the  pharyn- 
geal  muscles,  of  the  palate,  of  the  larynx  and  some  of  the  skeletal 
muscles,  nephritis  with  edema,  etc.).  Gabriel,  at  Neisser's  clinic, 


230  PASSIVE  IMMUNIZATION 

gave  4000  to  5000  units  every  five  days  for  four  weeks,  in  severe 
cases.  At  the  Berlin  Charite  four-fifths  of  the  cases  require  from 
1500  to  4500  units;  in  the  remainder  9000  to  18,000  are  common 
amounts,  and  in  the  severest  cases  30,000  to  65,000  have  been  used. 
Above  all,  then,  the  physician  should  not  despair  in  the  face  of  a 
grave  case,  but  use  the  antitoxin  systematically  until  the  child  is 
either  dead  or  out  of  danger.  The  same  rule  applies  in  the  man- 
agement of  those  cases  in  which  post-diphtheritic  paralyses  have 
occurred.  In  the  past  it  was  thought  that  antitoxin  would  be  of 
no  avail  in  such  cases,  but  in  the  light  of  more  recent  experience  it 
would  seem  that  here  also  much  good  may  come  from  the  systematic 
use  of  the  serum. 

The  question,  of  course,  suggests  itself,  why  antitoxin  should  still 
be  of  service  when  once  the  toxin  has  been  anchored  to  sensitive 
receptors;  but  I  would  recall  that  as  a  consequence  of  immunization, 
the  specificity  of  the  receptors  for  the  corresponding  antigen  may  be 
very  materially  increased  and  that  the  toxin  in  question  will  hence 
have  a  greater  affinity  for  the  antitoxin  furnished  by  the  horse 
than  for  the  sessile  receptors  of  the  patient.  Hence,  the  possibility 
exists,  theoretically  at  least,  that  an  active  antitoxin  may  be  able  to 
break  the  combination  between  the  toxin  and  the  patient's  recep- 
tors, and  our  clinical  experience  suggests  that  this  actually  occurs. 
To  effect  this  end,  however,  large  doses  are  evidently  necessary. 

Prophylactic  Dose. — For  prophylactic  purposes  a  dose  of  from 
500  to  1000  units  has  been  found  sufficient  to  afford  protection  for 
approximately  three  weeks  (see  curve  above).  Whether  or  not 
this  period  could  be  lengthened  by  the  administration  of  larger 
amounts  seems  doubtful,  in  view  of  the  fact  that  the  drop  in  the 
blood  content  of  antitoxin  which  takes  place  within  the  first  few 
days  of  its  injection  is  the  more  abrupt  the  larger  the  dose.  This 
will  be  understood  if  we  bear  in  mind  that  the  antitoxic  properties  of 
horse  serum  are  intimately  connected  with  its  globulins,  and  that 
these  are  alien  albumins  which  the  body  cannot  utilize  as  such  and 
which  it  accordingly  tries  to  destroy  as  soon  as  possible. 

Centra-indications  to  the  Use  of  Antitoxin. — In  view  of  the  fact  that 
a  small  number  of  people  are  hypersensitive  to  the  use  of  horse  serum 
to  such  a  degree  that  a  first  injection  even  may  be  followed  by  most 
alarming  symptoms,  and  in  rare  instances  by  death,  some  physicians 
have  of  late  hesitated  to  use  antitoxin  as  promptly  as  has  generally 


DIPHTHERIA  231 

been  urged.  Should  such  symptoms  develop,  it  is  recommended 
to  administer  atropin  and  adrenalin  hypodermically  and  to  resort 
to  artificial  respiration.  It  should  be  borne  in  mind,  however,  that 
actual  disaster  is  an  extreme  rarity  when  compared  with  the  innu- 
merable instances  in  which  antitoxin  is  used  without  any  untoward 
results,  and  that  the  danger  which  the  unprotected  patient  incurs 
from  the  diphtheria  is  infinitely  larger  than  that  which  would  likely 
follow  the  use  of  the  serum.  Unless,  therefore,  it  is  known  before- 
hand that  the  patient  is  hypersensitive  to  such  an  extreme  degree,  there 
should  be  no  hesitancy  on  the  part  of  the  physician  to  use  the  serum. 

It  would,  of  course,  be  ideal  if  some  method  could  be  worked 
out  which  would  enable  us  to  definitely  establish  the  existence  of 
abnormal  hypersensitiveness  before  the  injection,  but  as  yet  no 
such  method  exists.  In  some  instances  in  which  alarming  symptoms 
followed  the  injection  of  the  horse  serum  a  history  was  obtained 
that  the  patients  had  been  subject  to  asthmatic  attacks,  and  in 
some  of  these  such  attacks  were  brought  on  when  the  individual 
came  into  close  contact  with  horses.  It  would  accordingly  be  well 
to  inquire  into  this  point  before  the  injection  is  given,  and  possibly 
to  rule  out  from  the  treatment  all  those  in  whom  a  distinct  history 
of  asthma  is  obtained.  In  such  cases  antitoxin  derived  from  some 
other  animal  than  the  horse  could  probably  be  used  with  impunity, 
and  it  is  urgently  to  be  hoped  that  ere  long  the  manufacturers  will 
place  such  material  upon  the  market. 

This  could  then  also  be  employed  in  those  cases  in  which  horse 
serum  has  been  used  not  long  before,  and  in  which  we  would  hence 
have  reason  to  expect  the  development  of  a  sharp  attack  of  serum 
sickness.  The  nature  of  the  latter  we  have  already  discussed  before 
(Chapter  XI),  suffice  it  to  say  at  this  place  that  its  development 
cannot  be  regarded  as  a  contra- indication  to  the  use  of  the  serum, 
and  that  not  a  single  case  has  been  reported  in  which  the  serum  sick- 
ness in  itself  has  endangered  the  life  of  the  patient  or  caused  any 
permanent  damage  to  the  individual.  That  it  is  undesirable,  of 
course,  stands  to  reason,  and  as  the  liability  to  the  disease  increases 
to  a  certain  extent  with  the  amount  of  the  serum  employed,  it 
follows  that  sera  of  high  potency  in  small  bulk  are  generally  to 
be  preferred  to  larger  quantities  of  serum  of  low  antitoxic  content. 
As  the  blood  of  adults,  moreover,  has  been  found  to  contain  not 
inconsiderable  amounts  of  natural  diphtheria  antitoxin,  the  use  of 


232  PASSIVE  IMMUNIZATION 

horse  antitoxin  is  less  urgent  in  these  for  prophylactic  purposes  than 
in  children  and  can  indeed  often  be  neglected. 

The  Avoidance  of  Anaphylaxis  by  the  Production  of  Antianaphylaxis. 
—Of  late  the  suggestion  has  been  offered  that  it  may  be  possible 
to  produce  a  state  of  antianaphylaxis  (which  see)  by  injecting  the 
patient  with  a  small  quantity  (0.5  c.c.)  of  antitoxin  (sc.,  horse  serum) 
a  few  hours  before  the  principal  injection  is  made,  and  that  any 
dangers  arising  from  anaphylaxis  may  thus  be  minimized  or  alto- 
gether eliminated.  This  should  be  borne  in  mind  if  serum  treatment 
is  necessitated  in  a  patient  who  has  been  previously  injected  with 
horse  serum.  That  antianaphylaxis  may  indeed  develop  in  a  very 
short  time  following  the  introduction  of  serum  is  undoubted,  and 
Friedberger  has  lately  devised  an  apparatus  by  means  of  which  an 
intravenous  injection  of  serum  may  be  given  so  slowly  that  anti- 
anaphylaxis has  an  opportunity  to  develop  during  the  administration. 

Results. — If  now  we  come  to  study  the  effect  which  the  treat- 
ment of  diphtheria  with  antitoxin  has  had  upon  the  mortality  of  the 
disease,  it  is  apparent  from  a  survey  of  the  accompanying  table  that 
the  lowest  death-rate  will  be  obtained,  if  the  injections  can  be  given 
on  the  first  day,  and  that  the  mortality  percentage  increases  for 
every  day  that  the  treatment  is  delayed.  Taking  the  results  corre- 
sponding to  the  first  day  we  have  an  average  of  4.8  per  cent.  Further 
argument  than  this  should  be  unnecessary  to  convince  anyone 
that  in  the  use  of  antitoxin  we  now  have  a  weapon  in  the  face  of 
which  diphtheria  has  indeed  lost  its  terrors,  and  that  a  physician 
who  refuses  to  avail  himself  of  its  use  is  indeed  unfitted  to  practise  his 
profession. 


Author. 

Welch       .... 
Hilbert      .... 
Collective   report   of 
American  Pediatric 
Society 
Austrian     collective 
report    .... 
German     collective 
report    .... 

No.  of 

cases. 

1489 

2428 

5794 
1103 
9581 

Mortality 
per  cent. 

14.2 
18.3 

12.3 
12.6 
15.5 

First 
day. 
2.3 
2.2 

4.9 
8.0 
6.6 

Second 
day. 

8.1 
7.6 

7.4 
6.6 

8.3 

Third 
day. 

13.5 
17.1 

8.8 
9.8 
12.9 

Fourth 
day. 

19.0 
23.8 

20.7 
25.5 
17.0 

Fifth 
day. 

29.3 
33.9 

35.3 

28.8 
23.2 

Sixth 
day. 

34.1 
34.1 

30.7 

Later 
than, 
sixth. 

33.7 
38.2 

21.0 
26.9 

In  the  earlier  days  of  the  use  of  antitoxin  the  question  was  asked 
whether  the  lower  mortality  could  not  be  explained  on  the  assump- 


DIPHTHERIA  233 

tion  that  the  diphtheria  epidemic  which  was  then  prevailing  was 
of  an  unusually  mild  type.  We  know  as  a  matter  of  fact  that 
the  "virulence"  of  a  disease  undergoes  periodic  fluctuations,  so  that 
there  is  some  reason  in  such  a  suggestion.  But  even  so,  the  low 
mortality,  when  treatment  was  instituted  on  the  first  day,  which  was 
early  noted,  should  have  been  sufficient  to  dispose  of  this  possibility, 
for  up  to  that  time  no  treatment  that  had  been  previously  in  use 
could  boast  of  such  a  result.  But  aside  from  this  there  are  many 
other  observations  which  prove  beyond  a  shadow  of  a  doubt  that  the 
low  general  mortality  from  diphtheria  is  really  due  to  the  use  of 
antitoxin  and  not  to  accidental  factors.  At  the  Blegdam  Hospital 
of  Copenhagen  during  an  entire  year  all  diphtheria  patients  admitted 
on  alternate  days  were  thus  treated  with  antitoxin,  while  those 
entering  on  the  intervening  days  were  given  no  serum.  The  result 
was  the  following: 

Of  204  cases  without  croup  treated  with  serum  5  died,  giving  a 
mortality  of  2  per  cent. 

Of  210  cases  without  croup  treated  without  serum  14  died,  giving 
a  mortality  of  7  per  cent. 

Of  35  cases  with  croup  treated  with  serum  3  died,  giving  a  mor- 
tality of  8  per  cent. 

Of  43  cases  with  croup  treated  without  serum  15  died,  giving  a 
mortality  of  35  per  cent. 

Evidence  of  the  same  kind  is  afforded  by  the  observation  that 
during  the  year  1894  in  Heubner's  clinic  the  mortality  had  been 
lowered  to  23.08  through  the  use  of  antitoxin,  while  in  another 
hospital  in  the  same  city  where  no  antitoxin  was  as  yet  available, 
the  death-rate  was  43.36  per  cent.  Korte  further  reports  that  in  the 
days  preceding  the  introduction  of  the  serum  the  death-rate  among 
the  tracheotomized  children  in  his  clinic  was  77.5  and  subsequently 
52.4.  Similar  figures  were  obtained  by  Siegert  in  his  collective 
report  based  upon  an  analysis  of  30,369  operated  cases  of  diphtheritic 
larynx  stenosis;  of  these  17,499  belonged  to  the  pre-serum  time 
and  furnished  a  death-rate  of  60.38  per  cent.,  as  contrasted  with  a 
mortality  of  36.32  among  12,870  cases  that  had  been  treated  with 
antitoxin.  These  figures  speak  for  themselves. 

The  question,  of  course,  suggests  itself,  whether  it  should  not  be 
possible  to  abolish  the  death-rate  from  diphtheria  altogether,  if 
once  all  cases  could  be  treated  with  antitoxin  on  the  first  day  of  the 


234  PASSIVE  IMMUNIZATION 

disease.  As  a  matter  of  fact  there  are  physicians  who  have  not  a 
single  death  to  record  among  just  such  cases,  even  though  their 
experience  is  based  upon  a  fairly  large  number  of  observations. 
Nevertheless,  there  are  instances  where  the  injections  have  been 
started  in  time  and  in  which  death  nevertheless  occurred  (see  table 
above).  Whether  any  of  these  could  have  been  saved  by  injecting 
the  antitoxin  intravenously  or  by  using  larger  doses  is  now,  of  course, 
impossible  to  say,  but  the  possibility  unquestionably  exists.  But 
even  so  we  should  remember  that  our  serum  is  after  all  purely  anti- 
toxic in  character,  and  that  unless  the  body  can  successfully  destroy 
the  infecting  organisms  the  battle  may  yet  be  lost,  and  it  is  this 
factor  which  may  be  responsible  for  the  number  of  deaths  that  yet 
occur,  even  though  the  antitoxin  be  used  at  the  very  start.  To 
overcome  this  possible  obstacle  to  a  zero  mortality  it  would  be 
tempting  to  use  a  corresponding  vaccine  simultaneously  with  the 
antitoxin.  This  has  indeed  been  advocated  by  several  investigators 
and  deserves  serious  consideration.  Petruschky  records  that  he 
has  succeeded  in  freeing  bacillus  carriers  in  this  way  of  their 
dangerous  guests. 

TETANUS 

The  preparation  and  titration  of  tetanus  antitoxin  is  based  upon 
practically  the  same  principles  as  that  of  diphtheria  antitoxin,  which 
we  have  considered  in  some  detail  in  the  foregoing  section.  The 
standards  employed  in  Germany  are  the  following: 

One  unit  of  toxin  is  that  quantity  which  is  capable  of  killing 
4,000,000  white  mice  (of  an  average  weight  of  10  grams  each)  within 
four  or  five  days  with  the  characteristic  symptoms  of  tetanus. 

A  toxin  solution  of  such  strength  that  1  c.c.  contains  one  unit  of 
toxin  is  designated  as  normal  toxin. 

One  unit  of  antitoxin  is  that  quantity  which  will  protect  a  mouse 
weighing  10  grams  against  4,000,000  fatal  doses  of  toxin,  when 
injected  subcutaneously. 

A  normal  antitoxic  serum  is  one  of  which  1  c.c.  contains  one  unit 
of  antitoxin. 

In  the  United  States  an  official  standard  unfortunately  does  not 
yet  exist,  and  as  the  standards  of  the  different  manufacturers  are 
not  alike,  physicians  are  practically  obliged  to  express  their  dosage 
in  terms  of  cubic  centimeters  rather  than  in  antitoxin  units. 


TETANUS  235 

Von  Behring's  fluid  antitoxin  is  marketed  in  100  unit  (10  c.c.) 
and  20  unit  (2  c.c.)  doses;  in  addition  to  this  a  solid  antitoxin,  of 
which  20  units  represent  a  dose,  is  also  available. 

Dosage  and  Uses. — For  prophylactic  purposes,  20  units  (2  c.c.) 
should  be  injected  about  the  site  of  injury,  and  if  large  nerve  trunks 
have  been  exposed,  in  part  into  their  substance:  the  idea  being  to 
bind  the  toxin  which  is  formed  about  the  point  of  infection,  before 
it  leaves  this  district,  which  takes  place  along  the  lymphatics  and 
the  nerve  fibers.  At  the  same  time,  it  is  recommended  to  give 
a  subcutaneous  injection  of  100  additional  units  (10  c.c.)  at  an 
indifferent  point,  and  to  repeat  the  dose  within  six  to  eight  weeks, 
as  the  immunity  which  is  afforded  only  lasts  for  that  length  of  time. 

When  symptoms  of  tetanus  already  exist,  very  little  is  to  be 
expected  from  the  use  of  the  antitoxin  for  the  reason  that  these 
symptoms  indicate  that  a  union  with  sensitive  receptors  (in  the 
central  nervous  system)  has  already  occurred,  and  that  the  anti- 
toxin cannot  penetrate  to  those  points  from  intact  bloodvessels. 
Neither  the  subcutaneous  nor  the  intravenous  route  hence  offers 
much  hope  of  a  satisfactory  result.  The  attempt  has  accordingly 
been  made  to  bring  the  material  into  immediate  contact  with  the 
central  nervous  structures,  by  intraneural  injections,  through  intra- 
cerebral  injections  and  by  its  introduction  into  the  subarachnoid 
space.  The  intracerebral  method  is  to  be  deprecated  altogether, 
as  the  death-rate  following  its  use  has  been  exceedingly  high.  More 
appropriate  is  the  intraneural  route,  to  which  end  the  larger  nerve 
trunks,  along  which  absorption  has  likely  taken  place,  must  be 
exposed  and  injected  at  different  points  in  their  course.  Unfortu- 
nately, not  much  serum  can  be  introduced  in  this  manner,  and  it  is 
natural  that  the  patient  should  subsequently  suffer  a  good  deal  from 
the  resulting  neuritis.  By  the  subdural  route,  on  the  other  hand,  it 
is  easy  to  introduce  large  quantities  of  serum,  and  as  Stintzing  and 
Kiister  have  already  demonstrated  that  the  cerebrospinal  fluid 
usually  contains  a  considerable  amount  of  toxin  in  human  tetanus, 
this  method  of  treatment  seems  rational  and  likely  to  do  good  so 
long  as  recovery  is  at  all  possible,  i.  e.,  so  long  as  the  union  between 
toxin  and  the  sensitive  receptors  is  still  capable  of  being  broken. 
It  is  recommended  to  tap  the  subarachnoid  space  in  the  usual  manner, 
to  allow  as  much  of  the  meningeal  fluid  to  escape  as  possible,  care 
being  taken,  however,  not  to  let  the  pressure  fall  too  low,  and  then  to 


236  PASSIVE  IMMUNIZATION 

slowly  inject  an  equivalent  volume  of  serum  (10  to  20  c.c.)  at  a  rate 
of  about  2  c.c.  per  minute.  According  to  the  requirements  of  the 
case,  this  may  be  repeated  several  times  within  the  same  twenty- 
four  hours,  and  continued  on  the  following  days. 

As  antitoxin  treatment  in  tetanus  can  be  expected  to  do  good 
only  so  long  as  the  toxin  has  not  combined  with  the  sensitive  recep- 
tors of  the  central  nervous  system  (barring  those  exceptional  cases 
where  this  union  can  still  be  broken),  and  so  long  as  it  can  be  readily 
reached  by  the  antitoxin,  i.  e.,  before  it  has  begun  its  travel  along 
the  axis-cylinders  of  the  affected  nerves,  it  follows  that  its  use  must 
be  largely  limited  to  prophylactic  purposes.  As  the  treatment,  how- 
ever, is  of  signal  value,  when  employed  to  this  end,  the  practitioner 
should  resort  to  its  use  in  all  those  injuries  which  are  likely  to  favor 
infection  with  tetanus  bacilli.  It  is  hence  recommended  in  con- 
nection with  all  wounds  which  have  been  contaminated  with  earth, 
manure,  decomposing  vegetable  matter  of  any  kind,  particles  of 
clothing,  especially  in  puncture  wounds,  such  as  those  produced 
by  splinters  of  wood,  rusty  nails,  and  broken  crockery;  then  in  con- 
nection with  all  wounds  caused  by  exploding  fire-arms,  cartridges, 
fire-crackers,  rockets,  in  wounds  caused  by  unclean  instruments,  as 
on  battle  fields,  after  division  of  the  umbilical  cord,  removal  of  the 
placenta,  etc.  In  all  such  cases  the  use  of  tetanus  antitoxin  is  strongly 
to  be  advocated,  and  should  become  a  uniform  practice. 

When  once  tetanus  symptoms  have  developed,  very  little  can  be 
expected.  If  the  attempt  is  to  be  made,  however,  it  should  not  be 
delayed  unnecessarily,  and  the  subdural  route  chosen  by  preference. 
When  large  nerve  trunks  have  been  exposed,  intraneural  injections 
should  be  given  in  addition,  besides  which  subcutaneous  injections 
also  may  be  employed.  Intravenous  injections,  as  I  have  already 
pointed  out,  can  hardly  do  any  good. 

Results. — If  now  we  turn  to  an  analysis  of  the  results  which  the 
introduction  of  the  antitoxin  treatment  has  produced,  we  may 
practically  confine  our  attention  to  the  prophylactic  side  of  the  ques- 
tion. The  evidence  here  is  quite  conclusive  that  its  timely  use  may 
be  the  means  of  saving  many  lives.  In  our  own  country,  where  the 
anniversary  of  the  birth  of  the  nation's  independence  has  in  the  past 
been  annually  celebrated  by  a  tetanus  orgy,  the  death-rate  in  the 
absence  of  prophylactic  treatment  has  been  perfectly  appalling. 
Liell,  in  an  analysis  of  350  cases,  thus  reports  that  of  this  number 


TETANUS  237 

only  seven  recovered  (mortality  98  per  cent.),  of  which  five  had 
received  the  prophylactic  treatment.  Scherk  then  mentions  that  of 
591  cases  of  Fourth-of-July  injuries  which  received  prophylactic 
injections  of  antitoxin,  not  a  single  one  developed  the  disease. 
Equally  convincing  are  the  reports  from  certain  hospitals,  in  which 
ant  it  et  anus  injections  are  given  as  a  matter  of  routine  in  all  cases 
where  contamination  of  wounds  with  dirt  from  the  street  has  occurred 
and  where  the  disease  is  under  these  conditions  hardly  ever  seen. 

While  tetanus  is  a  fairly  common  malady  in  the  province  of 
Pommern  it  has  thus  been  noted  at  the  surgical  clinic  of  Greifswald, 
where  the  prophylactic  treatment  of  all  primary  injuries  has  been 
carried  out  for  a  number  of  years,  that  tetanus  among  the  injected 
is  practically  unheard  of,  while  it  is  common  enough  among  patients 
that  are  sent  in  from  the  surrounding  districts  where  this  treatment 
is  not  in  use.  In  Indo-China  further,  where  formerly  one-fifth  of  all 
newborn  children  succumbed  to  tetanus  of  umbilical  origin,  Calmette 
found  that  the  administration  of  the  dried  preparation  to  the  stump 
of  the  umbilicus  was  sufficient  to  prevent  the  outbreak  of  the  malady. 
Quite  suggestive  also  are  the  results  which  have  been  obtained  in 
veterinary  practice.  Nocard  thus  reports  that  in  a  certain  quarter 
of  Paris  where  tetanus  was  exceedingly  common  among  horses,  not 
a  single  case  developed  among  2727  injected  animals  concerning 
which  he  received  reports,  and  of  which  2300  had  been  castrated; 
while  during  the  same  period  of  time  there  occurred  259  cases  among 
those  that  had  not  been  protected. 

Evidence  of  this  sort  is  now  so  abundant  that  the  importance,  nay 
the  necessity,  of  prophylactic  treatment  in  the  injured,  where  there 
is  the  slightest  reason  for  anticipating  the  possible  development 
of  tetanus,  cannot  be  too  strongly  urged.  When  a  physician  nowa- 
days quietly  dresses  an  extensive  scalp  wound  of  the  head,  which 
has  been  freely  contaminated  with  manure,  and  does  not  give  his 
patient  the  benefit  of  a  prophylactic  injection  of  tetanus  antitoxin 
his  negligence  is  certainly  but  little  short  of  criminal. 

The  question  may,  of  course,  be  asked,  whether  tetanus  never 
develops  if  an  early  injection  of  antitoxin  be  given.  While  we  must 
admit  that  the  protection  is  not  absolute,  the  fact  remains  that  if 
tetanus  does  occur  under  such  conditions  its  course  is  very  mild. 
Kiister  thus  mentions  a  case  where  infection  occurred  accidentally 
in  a  laboratory  with  a  highly  virulent  culture,  and  where,  in  spite 


238  PASSIVE  IMMUNIZATION 

of  prophylactic  treatment,  tetanus  developed  on  the  sixth  day.  In 
such  a  case  ordinarily  death  would  unquestionably  have  followed, 
but,  as  it  was,  the  patient  had  an  uncommonly  mild  attack  which 
resulted  in  recovery. 

Regarding  the  effect  of  the  antitoxin  treatment  upon  the  malady 
when  once  this  has  developed,  very  little  need  be  said.  If  we  rule 
out  from  our  consideration  all  those  cases  in  which  the  first  symp- 
toms have  developed  after  nine  days  or  still  later,  we  may  say  that 
death  will  result  no  matter  whether  the  patient  is  injected  or  not. 
In  the  case  of  the  remainder,  we  must  remember  that  the  patient's 
chances  are  the  better  the  longer  the  period  of  incubation,  so  that 
the  conclusion  is  not  necessarily  warrantable  that  recovery  has 
taken  place  in  such  cases  because  of  the  injection.  The  best  that 
we  can  say  is  that  the  treatment  may  possibly  help,  but  that  we 
cannot  always  logically  attribute  recovery  to  the  treatment.  It 
should  be  tried,  but  not  too  much  should  be  expected. 


DYSENTERY 

While  the  attempts  at  prophylactic  vaccination  against  infection 
with  the  Shiga-Kruse  bacillus  have  not  led  to  very  satisfactory 
results  (see  p.  200),  there  is  evidence  to  show  that  the  use  of  the 
corresponding  antiserum  exerts  a  beneficial  influence  upon  the  course 
of  the  malady,  when  this  has  once  developed.  Regarding  the  mode 
of  action  of  the  antisera  which  were  first  prepared  by  Shiga  and 
Kruse,  there  has  been  some  controversy,  it  having  been  thought  at 
first  that  their  effect  was  essentially  bacteriolytic  in  nature.  Subse- 
quently, however,  when  it  was  shown  by  Kraus  and  Doerr  that  the 
dysentery  bacillus  produces  a  true  toxin,  and  that  the  same  effect 
could  be  obtained  with  an  antiserum,  produced  with  this  as  antigen, 
the  conclusion  naturally  suggested  itself  that  the  beneficial  effects 
reached  with  the  older  preparations,  where  unfiltered  cultures  includ- 
ing the  bodies  of  the  bacilli  represented  the  antigen,  were  probably 
also  owing  to  contained  antitoxins. 

Preparation. — The  preparation  of  antidysentery  serum  is  con- 
ducted along  similar  lines  as  that  of  the  other  sera,  which  are  used 
for  passive  immunization,  horses  being  employed  as  the  antibody 
producers.  As  in  the  immunization  against  diphtheria  and  tetanus 


DYSENTERY  239 

toxin  a  basic  (Grund)  immunity  is  first  established  by  injecting  a 
certain  quantity  of  antiserum  together  with,  or  twenty-four  hours 
preceding,  the  introduction  of  the  toxin,  or  the  toxin  cultures,  after 
which  the  process  is  continued  with  these  alone. 

Dosage  and  Uses. — The  serum  which  is  used  for  curative  purposes 
in  Vienna  is  of  such  strength  that  0.1  c.c.  at  most  will  protect  a 
rabbit  weighing  1000  grams  against  a  separate  though  simultaneous 
intravenous  injection  of  a  single  lethal  dose  of  the  toxin.  The 
curative  dose  of  such  a  serum  for  the  human  being  varies  between 
10  and  20  c.c.,  which  may  be  repeated  several  times  in  severe  cases. 
In  extreme  cases  the  French  observers  have  used  as  much  as  80  to 
100  c.c.  on  the  first  day,  and  have  repeated  this  on  the  following 
days.  In  three  instances  240,  380,  and  1080  c.c.  were  injected 
altogether,  i.  e.,  doses  which  seem  unwarrantably  and  unnecessarily 
large,  if  an  active  serum  was  really  at  hand.  After  the  disease 
comes  under  control,  as  is  evidenced  by  a  diminution  in  the  number 
of  the  stools,  smaller  doses  may  be  given  during  the  following  days. 

For  prophylactic  purposes  the  same  dosage  (10  to  20  c.c.)  is 
recommended,  and  it  is  further  advised  to  repeat  the  injections 
after  two  or  three  weeks,  as  the  protection  only  lasts  a  short  time.  As 
the  different  manufacturers  do  not  employ  the  same  standards  the 
practitioner  must  use  the  serum  in  accordance  with  the  printed 
directions  which  accompany  the  individual  package. 

Injections. — The  injections  are  given  subcutaneously  in  the  usual 
districts.  As  the  Shiga-Kruse  strains  alone  are  toxin  producers, 
while  the  Flexner  type  does  not  belong  to  this  order,  and  as  the 
serum  corresponding  to  the  former  is  markedly  specific  in  its  action, 
it  is  advisable  to  ascertain  at  the  time  of  an  epidemic  whether  the 
infection  is  actually  of  this  type.  Unless  this  is  done  it  would  not 
be  warrantable  to  ascribe  a  lack  of  action  to  the  serum  when  no 
effect  is  observed. 

Results. — Regarding  the  results  which  have  been  obtained  with 
the  serum  in  question,  it  seems  that  the  treatment  is  actually  quite 
useful  both  for  prophylactic  and  curative  purposes,  though  adequate 
statistics  are  not  yet  available.  More  convincing  than  mere  figures 
are  the  observations  which  have  been  made  at  the  sickbed,  by 
individual  observers,  all  of  whom  speak  quite  enthusiastically  of 
the  marked  effect  of  the  injections  upon  the  number  of  the  stools, 
which  frequently  drops  quite  suddenly  even  within  the  first  twenty- 


240  PASSIVE  IMMUNIZATION 

four  hours;  then  upon  the  pain  and  upon  the  general  condition  of 
the  patient.  Even  in  chronic  cases  much  benefit  may  be  expected. 
Veillard  and  Dopter  thus  mention  a  case  which  had  lasted  five 
months,  in  spite  of  the  most  varied  treatment,  where  recovery 
occurred  after  three  injections  of  serum. 

If  we  bear  in  mind  that,  next  to  typhoid  fever,  bacillary  dys- 
entery is  probably  the  most  formidable  common  disease  with  which 
military  surgeons  have  to  deal,  it  would  suggest  itself  that  in  times  of 
war,  or  when  large  bodies  of  men  are  concentrated  within  a  narrow 
compass  and  are  obliged  to  drink  water  of  unknown  quality,  pro- 
phylactic treatment  with  antidysentery  serum  might  prove  of  signal 
benefit. 

CHOLERA 

Although  a  number  of  different  attempts  have  been  made  to  pro- 
duce an  active  antiserum  for  the  treatment  of  Asiatic  cholera, 
nothing  of  real  value  has  as  yet  been  accomplished.  This  is  probably 
owing  to  the  fact  that  while  the  symptom  complex  of  cholera  is 
evidently  largely  the  result  of  an  intoxication,  the  toxins  in  question 
are  probably  only  in  small  part  true  toxins,  but  essentially  endotoxins 
against  which  antitoxins  are  produced  only  to  a  slight  extent,  if 
at  all. 

The  only  preparation  of  this  order  which  deserves  any  considera- 
tion, is  the  antiserum  of  Kraus,  in  the  production  of  which  the  El 
Tor  vibrio  was  used  as  antigen.  This  organism,  it  may  be  recalled, 
was  obtained  by  Gottschlich  in  1905  from  the  intestinal  contents  of 
pilgrims  who  had  died  at  El  Tor  from  dysentery,  and  is  not  identical 
with  the  true  cholera  vibrio,  but  evidently  very  closely  related  to 
it.  But  unlike  the  cholera  vibrio,  the  El  Tor  furnishes  a  true  toxin 
in  fairly  large  amount,  against  which  an  active  antitoxin  can  be 
obtained.  This  latter,  according  to  Kraus,  neutralizes  the  toxin 
of  true  cholera  as  well,  and  more  efficiently  than  the  antitoxin 
resulting  from  immunization  with  the  latter.  He  has  therefore 
recommended  it  for  the  treatment  of  Asiatic  cholera.  From  the 
reports  which  have  thus  far  been  obtained  it  is,  however,  scarcely 
possible  to  reach  a  definite  conclusion  regarding  its  value.  Ketscher 
and  Kernig  used  the  serum  in  119  severe  and  moderately  severe  cases, 
with  a  death-rate  of  58  per  cent,  in  those  who  had  received  subcu- 


PLAGUE  241 

taneous  injections,  and  one  of  50  per  cent,  when  used  intravenously; 
while  the  general  death-rate  among  the  non-injected  cases  was  63.4 
per  cent.  Other  observers  contrast  a  mortality  of  57.5  per  cent, 
among  the  treated  with  one  of  84.3  per  cent,  among  the  untreated 
cases.  The  verdict  among  those  who  have  had  experience  with  the 
serum  seems  to  have  been  that  the  serum  treatment  produced  a 
favorable  rather  than  an  unfavorable  impression,  which,  after  all, 
is  scanty  praise. 

Jegunoff  administered  the  serum  intravenously,  together  with 
physiological  salt  solution,  giving  140  c.c.  of  serum  with  500  to 
700  c.c.  of  saline  to  start  with,  and  a  second  injection  of  80  to 
120  c.c.  of  serum  within  seven  and  one-half  to  twenty-three  hours 
after  the  first. 

TYPHOID    FEVER 

While  antisera  against  typhoid  fever  have  been  proposed  by  a 
number  of  different  observers,  their  value  seems  to  be  so  problem- 
atical that  their  discussion  may  very  well  be  omitted  at  this  place. 


PLAGUE 

Against  plague  also  antisera  have  been  produced,  which  seem  to 
be  essentially  of  bactericidal  nature  (Yersin),  though  the  preparation 
of  Lustig  may  have  feeble  antitoxic  properties.  Both  the  serum  of 
Lustig  and  that  of  Yersin  have  been  tried  out  by  the  Plague  Com- 
mission of  India,  but  the  reports  are  not  very  encouraging.  Whether 
its  use  in  combination  with  vaccination  might  not  prove  of  greater 
value  than  vaccination  alone,  and  especially  in  persons  who  have 
been  actually  exposed  to  the  infection,  future  investigations  will 
have  to  decide,  but  w^ould,  a  priori,  seem  likely. 


BACTERIOLYTIC-BACTERIOTROPIC   IMMUNIZATION. 

Among  the  bacteriolytic-bacteriotropic  immune  sera  which  find 
employment  in  the  treatment  of  maladies  to  which  the  human  being 
is  subject,  the  most  important  are  those  which  are  directed  against 
16 


242  PASSIVE  IMMUNIZATION 

infections  with  the  pyogenic  cocci,  viz.,  the  meningococcus,  the 
streptococcus,  the  pneumococcus,  the  gonococcus,  and  the  staphylo- 
coccus.  Of  these  the  antimeningococcus  serum  is,  however,  prac- 
tically the  only  one  with  which  notable  curative  results  have  been 
obtained.  It  will  accordingly  be  considered  in  some  detail,  while  the 
remainder  need  not  occupy  our  attention  to  any  great  degree. 


MENINGOCOCCUS   MENINGITIS 

Attempts  to  produce  an  antiserum  for  the  treatment  of  meningo- 
coccus meningitis  in  the  human  being  have  notably  been  made  by 
Flexner  and  Jobbling,  Kolle  and  Wassermann,  and  Jochmann,  and 
it  may  be  said  that  the  efforts  of  all  these  investigators  have  been 
crowned  with  a  great  degree  of  success.  From  the  therapeutic 
standpoint  very  little  difference  indeed  appears  to  exist  in  the 
efficacy  of  the  three  preparations  in  question,  but  there  is  still  a 
good  deal  of  difference  of  opinion  in  regard  to  their  mode  of  action. 
All  three  contain  agglutinins,  precipitins,  complement  binding  anti- 
bodies, bacteriolysins,  bacteriotropins,  and  possibly  also  some  anti- 
toxins. From  the  different  accounts  that  have  been  given,  the  con- 
clusion suggests  itself  that  while  antitoxins  may  possibly  play  a  role, 
this  is  unquestionably  of  minor  importance  when  compared  with 
the  marked  inhibitory  effect  which  the  serum  exercises  upon  the 
multiplication  of  the  organisms,  and  to  its  manifest  bacteriotropic 
action,  as  evidenced  by  increased  phagocytosis. 

Flexner  thus  records  that  in  two  children  who  had  received  sub- 
dural  injections  of  his  serum,  scarcely  any  extracellular  diplococci 
could  be  found  after  the  first  treatment,  while  the  number  of  intra- 
cellular  cocci  was  much  reduced,  and  that  cultures  could  no  longer 
be  secured,  even  though  the  free  forms  had  not  yet  disappeared 
altogether. 

Flexner  suggests  that  the  phagocytic  digestion  not  only  pre- 
vents further  multiplication  of  the  diplococcus,  but  also  that  it 
detoxicates  the  endotoxin  by  reducing  it  to  simpler  and  non-toxic 
or  less  toxic  compounds. 

That  bacteriolysins  per  se,  however,  may  also  play  a  role  is  sug- 
gested by  the  observation  that  in  a  few  instances  in  which  the 
antiserum  was  injected  into  the  spinal  canal  of  monkeys  infected 


MENINGOCOCCUS  MENINGITIS  243 

with  the  diplococcus  the  microorganisms  disappeared  without 
marked  phagocytosis,  though  more  slowly  than  in  the  cases  in  which 
outpouring  of  leukocytes  was  considerable. 

Preparation  of  the  Antimeningococcus  Serum  (according  to  Flexner 
and  Jobling) . — Horses  are  first  injected  subcutaneously  with  cultures 
of  the  diplococcus  that  have  been  heated  for  thirty  minutes  at 
60°  C.,  as  many  different  strains  being  used  collectively,  as  possible, 
so  as  to  give  rise  to  a  polyvalent  serum.  As  first  dose  the  equivalent 
of  a  quarter  surface  test-tube  growth  on  sheep-serum  agar  is  recom- 
mended. At  each  subsequent  injection  the  dose  is  doubled  until 
an  amount  equal  to  four  test-tube  growths  is  given  at  intervals  of 
five  to  seven  days. 

In  the  earlier  work  of  Flexner  and  Jobling,  intravenous  inoculations 
were  then  substituted  for  the  subcutaneous,  beginning  with  one  dose 
of  living  diplococci,  the  dose  being  progressively  increased  to  two, 
three,  five,  etc.,  oeses,  then  to  one-half,  three-quarters,  one,  two, 
etc.,  agar  slant  cultures,  and  finally  to  one  and  a  half  bottles  (12  oz. 
Blake)  of  surface  growth.  As  the  larger  injections  caused  very 
severe  reactions  and  alarming  symptoms,  they  were  discontinued, 
and  subcutaneous  and  intravenous  injections  of  autolysates1  sub- 
stituted, the  dose  being  gradually  increased  from  1  to  3  c.c.  and 
given  about  a  week  apart.  Since  the  intravenous  injections  of  the 
autolysates,  however,  likewise  produced  quite  serious  symptoms, 
they  also  were  abandoned,  and  at  present  subcutaneous  injections 
only  are  recommended  for  the  whole  process  of  immunization, 
living  diplococci  and  autolysates  being  used  alternately  at  intervals 
of  a  week.  The  maximum  dose  of  living  organisms  and  of  the  auto- 
lysates is  one  and  one-half  bottles. 

The  process  of  immunization  in  Flexner's  horses  was  continued 
for  a  year  or  longer,  before  any  of  the  serum  was  used  for  purposes 
of  treatment. 

Standardization. — Unfortunately  no  method  is  at  present  avail- 
able by  which  the  curative  or  protective  effect  of  the  antimeningo- 
coccus  serum  can  be  gauged  other  than  by  actual  trial.  Kolle 
and  Wassermann  attempted  to  standardize  their  serum  on  the  basis 
of  its  content  in  complement  binding  antibodies,  in  the  belief  that 
these  were  identical  with  the  bacteriolytic  amboceptors.  This  idea, 

1  Meningococci  which  have  been  allowed  to  undergo  self-digestion. 


244  PASSIVE  IMMUNIZATION 

however,  has  been  shown  to  be  erroneous,  and  the  method  is  from 
this  standpoint  therefore  inapplicable.  Other  investigators  have 
suggested  to  use  the  bactericidal  power  of  the  serum  in  mw  as 
indicator  of  its  therapeutic  properties.  The  values  which  can  thus 
be  obtained  are,  however,  approximative  at  best,  and  the  same  is 
to  be  said  regarding  Neuf eld's  suggestion  to  gauge  its  strength  by  a 
determination  of  its  bacteriotropic  titer.  Kraus  and  Doerr,  in 
the  belief  that  the  efficiency  of  the  serum  depends  upon  its  content 
of  antitoxins,  suggest  its  standardization  upon  this  basis,  in  a  manner 
analogous  to  the  standardization  of  diphtheria  antitoxin,  but  it  is 
extremely  doubtful  whether  these  actually  play  an  important  role, 
and  the  suggestion  has  hence  not  met  with  favor.  Under  these 
circumstances  it  is  apparent  that  the  main  stress  must  be  placed 
upon  the  duration  of  the  immunizing  process,  possibly  coupled  with 
a  study  of  its  bactericidal  power  in  mw,  and  its  bacteriotropic 
effect. 

Dosage  and  Mode  of  Administration. — From  what  has  just  been 
said,  it  is  clear  that  the  dosage  of  the  serum  still  rests  upon  an 
empirical  basis.  As  initial  dose,  Flexner  recommends  an  injection  of 
30  c.c.,  which  may  be  repeated  every  twenty-four  hours  for  three  or 
four  days  or  longer.  All  injections  should  be  made  into  the  sub- 
arachnoid  space,  care  being  taken  that  the  serum  is  introduced  very 
slowly,  so  as  to  cause  no  symptoms  of  pressure.  It  is  hence  best  to 
allow  at  least  as  much  fluid  to  escape  as  it  is  desired  to  introduce. 

As  the  best  results  are  obtained  in  early  cases  (see  below)  every 
effort  should  be  made  to  reach  a  definite  diagnosis  as  soon  as  possible, 
and  to  this  end  spinal  puncture  is  practically  imperative.  If  this 
reveals  a  turbid  fluid  the  antiserum  may  be  injected  at  once,  the 
microscopic  and  bacteriological  examination  being  carried  out  later. 
If  this  should  prove  that  the  case  was  not  one  of  meningococcus 
meningitis,  no  harm  will  have  been  done,  while  in  the  event  of  a 
confirmatory  diagnosis,  valuable  time  will  have  been  gained.  The 
appearance  of  the  fluid  at  subsequent  examinations,  aside  from 
the  physical  condition  of  the  patient,  will  then  be  a  fairly  good 
index  as  to  the  necessity  of  repeating  the  injections.  So  long  as 
this  is  cloudy  further  treatment  is  needed.  All  in  all  it  is  better 
to  inject  too  much  and  too  often  than  too  little  and  too  infrequently. 
Late  in  the  disease,  however,  when  chronic  hydrocephalus  has 
developed,  the  treatment  is  useless. 


MENINGOCOCCUS  MENINGITIS  245 

The  subcutaneous  or  intravenous  use  of  the  serum  is  to  be  depre- 
cated, as  the  results  following  this  method  of  administration  are  no 
better  than  under  the  expectant  plan  of  treatment. 

One  question  of  great  practical  importance  which  has  arisen  in 
connection  with  the  serum  treatment  of  meningococcus  meningitis  is 
whether  any  danger  due  to  anaphylaxis  is  to  be  anticipated  from  the 
repeated  injections,  particularly  since  these  are  made  into  the  sub- 
arachnoid  space  and  since  Besredka  has  shown  that  the  direct  in- 
jection of  the  alien  serum  into  the  central  nervous  system  is  par- 
ticularly fatal  to  guinea-pigs.  So  far  as  we  can  tell,  this  danger  is 
really  a  negligible  quantity,  especially  as  the  daily  injections  in  the 
early  course  of  the  treatment  do  not  enter  into  consideration,  and 
the  patient  usually  is  beyond  the  need  of  serum  by  the  time  that 
anaphylactic  reactions  would  be  expected  to  occur.  But  even  in 
cases  where  the  injections  were  continued  into  this  period,  serious 
symptoms  have  not  been  observed. 

Results. — So  far  as  the  results  of  the  serum  treatment  upon  the 
course  of  the  disease  are  concerned,  we  have  sufficient  evidence  to 
show  that  through  its  introduction,  one  of  the  most  fatal  diseases, 
and  one  of  the  most  dangerous  in  its  late  effects,  even  in  cases  where 
recovery  has  occurred,  has  lost  some  of  its  terrors.  As  regards  its 
effect  upon  the  mortality,  much  depends  upon  the  time  at  which 
it  is  instituted.  Of  241  cases  which  had  thus  been  injected  with 
the  Flexner  serum  during  the  first  three  days  of  the  malady,  only 
25.3  per  cent,  died,  while  a  delay  of  from  one  to  four  days  beyond 
this  period  increased  the  death  rate  to  27.8  per  cent.,  and  a  still 
further  delay  to  42.1  per  cent.  The  general  death-rate  of  712  treated 
cases  was  31.4  per  cent.,  as  contrasted  with  the  usual  mortality  of 
from  53  to  90  per  cent.  By  eliminating  all  those  cases  where  the 
patients  were  first  seen  in  an  already  hopeless  condition,  but  injected 
nevertheless,  Flexner  calculated  an  average  mortality  of  25.4  per 
cent.  Similar  results  have  been  reached  with  the  sera  prepared  by 
Wassermann,  Jochmann,  and  Dopter.  The  latter  claims  an  average 
mortality  of  only  16.47  per  cent.  (402  cases)  for  his  serum,  as  con- 
trasted with  one  of  65  per  cent,  in  untreated  cases;  Schone  one  of 
27  per  cent,  for  Jochmann's  serum  (in  a  relatively  small  number 
of  cases),  and  Dopter  one  of  18.35  per  cent.  (158  cases)  for  that  of 
Wassermann. 

The  immediate  effect  upon  the  malady  is  also  quite  favorable; 


246  PASSIVE  IMMUNIZATION 

usually  within  twenty-four  to  forty-eight  hours  there  is  definite 
improvement,  as  evidenced  by  a  return  to  consciousness,  disappear- 
ance of  delirium,  diminution  of  the  general  hypersensibility ,  etc. 
The  duration  of  the  disease  is  shortened  to  eight  to  twelve  days, 
as  contrasted  with  five  weeks  or  longer,  which  is  the  rule  in  fully 
one-half  of  the  cases  that  end  in  recovery,  in  the  absence  of  serum 
treatment. 

In  conclusion  it  would  seem  that  late  effects  of  the  malady  are 
only  exceptionally  observed;  mental  disorder,  paralysis  and  blind- 
ness in  particular  are  only  rarely  seen. 

We  may  accordingly  look  with  pride  and  satisfaction  upon  the 
antimeningitis  work  as  one  of  the  brightest  pages  in  the  history 
of  serology. 

STREPTOCOCCUS   INFECTIONS 

Since  the  days  when  v.  Behring  first  came  forward  with  the 
announcement  that  it  is  possible  with  the  serum  of  an  animal  that 
has  been  immunized  against  the  corresponding  toxin,  not  only  to 
protect  individuals  against  diphtheria,  but  even  to  cure  the  disease 
after  this  has  once  developed,  attempt  after  attempt  has  been  made 
to  produce  an  effective  antiserum  also  against  streptococcus  infec- 
tions. But  as  yet  the  problem  has  not  been  solved.  Much  work 
of  value  has  been  accomplished,  but  still  more  remains  to  be  done. 
That  it  is  possible  to  protect  animals  against  a  fatal  infection  with 
streptococci  by  means  of  a  corresponding  antiserum  had  been  shown 
by  v.  Behring  himself  in  1892,  and  shortly  after  a  number  of  French 
observers  attempted  to  influence  the  infection  in  the  human  being 
also  in  a  similar  manner. 

The  most  noteworthy  of  these  early  attempts  is  intimately  con- 
nected with  the  name  of  Marmoreck.  This  investigator,  believing 
in  the  unity  of  practically  all  the  different  types  of  streptococci 
which  are  pathogenic  for  man,  succeeded  in  increasing  the  virulence 
of  an  angina  strain  by  animal  passage  to  such  a  degree  that 
0.000000001  c.c.  was  sufficient  to  kill  a  rabbit  with  acute  symptoms. 
With  this  strain  he  immunized  horses  and  sheep  and  then  recom- 
mended the  resulting  antiserum,  which  is  thus  a  monovalent  serum, 
for  the  treatment  of  all  forms  of  streptococcus  infections  occurring 
in  the  human  being.  The  results,  however,  were  practically  nil. 


STREPTOCOCCUS  INFECTIONS  247 

If  we  come  to  investigate  the  reasons  which  may  be  responsible 
for  this  want  of  action,  different  possibilities  suggest  themselves. 
On  the  one  hand  it  is  conceivable  that  the  identity  of  the  different 
strains  is  only  apparent  and  that  Marmoreck's  serum  was  inactive 
merely  because  it  was  monovalent,  i.  e.,  because  it  had  been  produced 
with  but  a  single  strain.  If  this  were  so  it  would  evidently  be 
necessary  to  prepare  a  polyvalent  antiserum,  i.  e.,  to  immunize 
animals  with  as  many  different  strains  as  possible,  and  to  use  the 
resultant  product.  Or,  one  might  imagine  that  in  consequence  of 
animal  passage,  to  increase  the  virulence  of  a  different  strain,  the 
organism  could  become  so  altered  in  its  biological  properties  that  its 
virulence  for  the  human  being  would  be  diminished  or  lost,  and  that 
the  corresponding  antiserum,  though  active  for  the  animals  through 
which  the  passage  had  been  conducted,  might  still  be  inactive  in 
the  human  being.  In  such  an  event,  animal  passage  would  have  to 
be  omitted,  and  a  monovalent  or  polyvalent  serum  prepared  by 
immunizing  directly  with  strains  that  had  been  obtained  from  human 
beings  (sc.,  with  cultures  made  from  human  sources  only). 

Both  possibilities  have  indeed  been  considered  and  practically 
tested.  Denys  and  Van  der  Velde  thus  prepared  a  polyvalent 
serum  from  a  number  of  different  strains,  whose  virulence  had  been 
further  increased  by  animal  passage,  but  this  serum  also  has  fallen 
into  oblivion,  which  suggests  that  subsequent  investigations  did  not 
support  the  favorable  reports  which  first  followed  its  introduction. 
Tavel,  Krumbein,  and  Paltauf,  on  the  other  hand,  prepared  polyva- 
lent sera  from  different  human  strains  without  animal  passage, 
and  Menzer  and  Moser  monovalent  strains  which  had  likewise  not 
been  passed  through  animals;  while  Aronson  attempted  a  combined 
procedure  making  use  of  passed  and  unpassed  organisms  conjointly, 
both  in  the  form  of  monovalent  and  polyvalent  preparations.  At 
the  present  time  practically  all  these  products  are  in  use,  and  while 
they  are  unquestionably  efficacious  in  the  animal  experiment,  the 
clinical  evidence  is  still  rather  against  than  in  favor  of  their  real 
value.  This  suggests  the  possibility,  of  course,  that  clinicians  may 
not  apply  the  sera  as  promptly  in  streptococcus  infections  as  is 
done  in  diphtheria,  and  as  a  matter  of  fact  there  is  a  good  deal  of 
truth  in  this  criticism.  That  this  factor  may  actually  be  one  of 
moment  is  suggested  by  the  fact  that  the  best  results  have  thus  far 
been  obtained  in  scarlatina,  where  the  diagnosis  is  reached  at  an 


248  PASSIVE  IMMUNIZATION 

early  date,  and  where  the  serum  can  be  conveniently  and  system- 
atically tested.  In  the  other  streptococcus  infections  the  bacterio- 
logical diagnosis  is  frequently  not  made  at  all,  or  it  is  delayed  until 
it  would  seem  unreasonable  to  expect  any  favorable  result.  Here, 
as  elsewhere,  in  serum  therapy,  the  clinician  should  bear  in  mind 
that  the  greatest  good  will  only  be  accomplished,  if  the  various  anti- 
sera  are  used  early,  in  sufficient  quantity,  and  usually  in  repeated 
doses. 

Mode  of  Action. — Regarding  the  mode  of  action  of  the  various 
antistreptococcus  sera,  it  would  seem  that  this  is  to  a  great  extent 
bacteriotropic  in  character,  for  whereas  in  unprotected  animals 
an  intraperitoneal  inoculation  with  an  appropriate  number  of  organ- 
isms is  followed  by  a  relatively  insignificant  hyperleukocytosis  and 
phagocytosis,  while  the  organisms  multiply  without  any  very  evident 
restraint,  the  treated  animals  show  exactly  the  opposite  picture, 
i.  e.,  extensive  hyperleukocytosis  and  phagocytosis  without  evidence 
of  multiplication.  The  same  can  be  shown  outside  of  the  body, 
directly  under  the  microscope;  for  whereas  in  the  presence  of  normal 
serum,  washed  leukocytes  will  scarcely  take  up  any  virulent  strep- 
tococci, they  do  so  readily  when  in  contact  with  immune  serum. 

Whether  or  not  bacteriolytic  processes  also  play  a  role  in  the 
protection  of  the  animal  with  suitable  immune  sera  is  still  a  matter 
of  dispute.  Antitoxins,  on  the  other  hand,  certainly  are  not  present. 

Preparation  and  Standardization. — The  preparation  of  the  antistrep- 
tococcus sera  is  conducted  essentially  on  the  same  lines  as  that  of 
other  non-antitoxic  sera,  viz.,  by  starting  the  immunization  with 
small  doses  of  killed-off  cultures  and  progressively  increasing  the  dose, 
until  finally  full  virulent  living  organisms  are  injected.  At  the 
Serum  Institute  of  Vienna,  bouillon  cultures  of  from  two  to  eight 
days'  growth  are  used,  the  initial  dose  being  0.5  c.c.,  and  the  final 
one  varying  between  100  and  200  c.c.,  all  the  injections  being  given 
subcutaneously.  The  animals  are  not  bled  until  the  immunization 
has  been  continued  for  about  six  months.  The  serum  is  then  tested 
in  reference  to  its  bacteriological  purity  and  titer,  and  finally  put  up 
in  doses  of  50  and  100  c.c.  each,  without  any  preservative. 

In  making  up  the  polyvalent  antigen  for  immunization,  it  is  con- 
venient, even  though  all  the  other  strains  be  of  human  origin  and 
not  passed  through  animals,  to  introduce  one  strain  which  has  been 
so  treated  and  brought  to  a  high  degree  of  virulence,  for  mice  for 


STREPTOCOCCUS  INFECTIONS  249 

example,  so  as  to  have  an  approximative  indicator  at  least  for  the 
potency  of  the  antiserum,  this  being  then  standardized  against  that 
particular  "animalized"  strain.  At  the  same  institute  that  dose 
of  streptococci  which  will  kill  a  white  mouse  at  the  expiration  of  or 
just  preceding  the  end  of  four  days  is  designated  as  a  single  lethal 
dose;  but  in  testing  an  antiserum,  ten  times  this  amount  is  chosen 
as  the  dose  against  which  one  unit  of  antiserum  should  afford  pro- 
tection. A  simple  normal  serum  is  one  of  which  0.01  c.c.  will  afford 
this  degree  of  protection,  and  1  c.c.  of  such  a  serum  is  said  to  con- 
tain a  single  immunization  unit,  and  1  c.c.  will  accordingly  protect 
1000  mice  against  a  single  lethal  dose  each.  The  Vienna  serum,  as 
it  is  now  marketed,  contains  20  to  40  units  to  the  cubic  centimeter. 

Dosage  and  Uses. — Prophylactic  Doses. — For  prophylactic  pur- 
poses, antistreptococcus  serum  has  been  recommended  in  connection 
with  scarlatina  and  the  puerperal  process,  either  by  itself  or  in  combi- 
nation with  the  use  of  a  vaccine.  To  prepare  the  latter,  F.  Meyer 
suggests  that  a  bouillon  culture  of  a  corresponding  strain  be  obtained, 
centrifugalized,  the  sediment  washed  repeatedly  with  saline,  and 
finally  emulsified  with  a  quantity  of  0.5  per  cent,  carbolic  acid  in 
saline,  equal  to  the  volume  of  the  initial  culture.  The  resultant 
emulsion  is  killed  off  by  heating  for  six  hours  at  a  temperature  of 
65°  C.,  when  it  is  tested  bacteriologically  and  shaken  overnight  in  a 
shaking  machine.  This  constitutes  the  finished  vaccine,  which  does 
not  need  to  be  counted  out.  The  individual  in  question  receives  a 
serum  injection  of  20  c.c.,  and  at  intervals  of  three  days  increasing 
doses  of  the  vaccine  (0.1,  0.2,  0.4,  0.8,  and  1.6  c.c.). 

Curative  Dose. — For  curative  purposes  the  serum  has  been  used 
in  scarlatina,  in  severe  streptococcus  infections  of  the  throat,  in 
erysipelas,  in  puerperal  streptococcus  infections,  in  chronic  strepto- 
coccus infections  associated  with  tuberculosis  and  malignant  growths, 
in  streptococcus  endocarditis  and  arthritis,  etc.  In  scarlatina  the 
treatment  is  indicated  especially  in  those  cases  in  which  the  throat 
infection  is  at  all  severe,  or  in  which  the  initial  general  symptoms 
suggest  the  likelihood  of  a  severe  course.  In  cases  of  the  first  type 
the  injection  of  50  to  100  c.c.,  given  subcutaneously,  and  repeated 
once  or  twice,  if  necessary,  is  usually  sufficient,  while  in  severe 
systemic  infections,  when  the  blood  examination  frequently  shows 
the  presence  of  large  numbers  of  organisms,  still  larger  doses,  and 
repeated  even  more  frequently,  are  advocated.  In  cases  of  pro- 


250  PASSIVE  IMMUNIZATION 

tracted  sepsis,  vaccination  (see  above)  may  well  be  combined  with 
the  serum  treatment.  In  fulminating  cases,  where  blood  examina- 
tion reveals  the  presence  of  streptococci  already  within  a  few  hours 
of  the  first  appearance  of  symptoms,  nothing  short  of  an  intravenous 
injection  (50  c.c.)  should  be  tried,  and  it  would  seem  worth  while 
in  just  such  cases,  in  fact  in  all  the  more  severe  infections,  to  inject 
the  serum  diluted  with  normal  salt  solution,  as  has  been  suggested 
by  F.  Meyer,  or  to  follow  its  injection  with  a  subcutaneous  infusion 
of  500  c.c.  or  more. 

In  severe  streptococcus  anginas  the  dosage  is  essentially  the  same 
i.  e.,  50  c.c.,  given  subcutaneously  and  diluted,  if  desired,  the  dose 
being  repeated  in  accordance  with  the  urgency  of  the  symptoms, 
and  two  injections  a  day  given  if  necessary. 

In  erysipelas  the  use  of  the  serum  is  advocated  especially  in  cases 
affecting  the  head  and  neck,  as  also  in  migratory  cases,  while  the 
facial  type  of  the  disease  usually  does  well  with  ordinary  treatment. 
The  dosage  here  also  ranges  between  50  and  100  c.c.  according  to 
the  gravity  of  the  case. 

In  puerperal  infections  the  rule  should  be  to  use  the  serum  early 
or  not  at  all.  A  great  deal  of  valuable  time  is  here  often  lost  in 
waiting  to  ascertain  whether  the  infection  will  not  cure  itself.  The 
patient  should  receive  the  benefit  of  the  doubt,  no  matter  whether 
the  statistics  are  thereby  unduly  turned  in  favor  of  the  serum  or 
not.  Its  use  is  logical  and  should  be  resorted  to  in  every  case  where 
fever  develops  during  the  puerperal  period,  if  this  is  not  manifestly 
sapremic  in  character.  50  c.c.  given  subcutaneously  is  sufficient 
in  the  milder  cases,  while  in  the  presence  of  ominous  symptoms 
larger  doses  should  be  employed  (100  to  200  c.c.),  which  here,  also, 
may  be  suitably  combined  with  a  subcutaneous  infusion  of  saline 
(500  to  1000  c.c.).  In  urgent  cases  intravenous  injections  should 
be  made  (50  c.c.).  After  hysterectomy  it  is  recommended  to  give 
an  intraperitoneal  infusion  of  500  c.c.  of  serum  with  1000  c.c.  of 
saline,  the  operation  being  preceded  by  an  intravenous  injection 
of  100  c.c.  In  cases  which  have  become  chronic  the  serum  treatment 
should  be  combined  with  the  use  of  an  autogenous  streptococcus 
vaccine. 

In  the  chronic  infections  associated  with  endocarditis,  arthritis, 
tuberculosis,  and  carcinoma,  etc.,  much  smaller  doses  are  given,  viz., 
5  to  20  c.c.,  as  larger  amounts  are  apt  to  cause  an  aggravation  of 


STREPTOCOCCUS  INFECTIONS  251 

some  of  the  symptoms,  and  notably  temperature  disturbances  lasting 
for  sixteen  to  twenty-four  hours.  But  in  these  cases  more  good 
may,  cceteris  paribus,  be  expected  from  the  use  of  a  vaccine  which 
should,  if  possible,  be  autogenous,  than  from  the  serum.  (Both 
may,  however,  be  advantageously  combined.) 

Results. — Upon  surveying  the  literature  in  reference  to  the  cura- 
tive value  of  antistreptococcus  serum,  one  is  struck  with  the  fact 
that  while  diphtheria  antitoxin  is  generally  used  as  early  as  possible, 
the  antistreptococcus  serum  is  usually  resorted  to  too  late  and  in 
insufficient  amount.  The  result  is,  that  from  a  statistical  stand- 
point the  general  verdict  has  been  rather  unfavorable.  This  empha- 
sizes the  importance  that  immunization  treatment  in  hospital  work 
particularly  should  be  placed  in  the  hands  of  especially  trained 
men,  who  should  be  consulted  in  doubtful  cases.  My  belief  is  that 
then  and  only  then  immunotherapy  will  yield  its  best  results. 

As  I  have  already  indicated,  the  most  favorable  reports  have 
been  published  in  connection  with  the  use  of  the  serum  in  scarlatina. 
Escherich,  speaking  of  the  effect  of  the  Moser  serum,  remarks  that 
this  is  "zauberhaft"  (magic),  and  especially  so  when  used  early.  Of 
112  cases  which  had  been  injected  on  the  second  or  third  day  every 
one  recovered,  while  among  those  in  whom  the  treatment  had  been 
delayed  the  mortality  ranged  between  13  and  50  per  cent. 

In  erysipelas,  very  curiously,  the  least  favorable  results  have 
been  obtained;  in  the  migratory  forms,  however,  the  disease  usually 
comes  to  a  standstill  in  from  three  to  four  days.  The  facial  cases, 
of  course,  should  not  be  included  in  an  analysis  of  the  results,  as 
they  usually  do  well  without  serum  treatment.  In  puerperal  cases 
the  testimony  is  most  conflicting.  Some  observers,  such  as  Bumm, 
Peham,  and  Burkard,  thus  speak  quite  favorably  of  its  use  (when 
employed  early),  Burkard  reporting  50  cases,  of  which  twenty-nine 
were  pure  streptococcus  infections,  without  a  single  death,  while 
others  deny  having  seen  any  good  accomplished  whatever.  It  is 
in  these  very  cases  that  I  would  advocate  that  the  serum  treat- 
ment should  be  placed  in  the  hands  of  experts  who  shall  decide  how 
the  serum  is  to  be  given,  when  it  is  to  be  given,  and  how  much  is  to 
be  given.  That  even  then  there  will  be  unfavorable  results  also  is 
to  be  expected,  but  it  would  stand  to  reason  that  the  maximum 
amount  of  good  that  could  be  accomplished  would  be  obtained  under 
such  conditions. 


252  PASSIVE  IMMUNIZATION 

Regarding  the  value  of  the  serum  in  other  streptococcus  infections, 
too  little  is  as  yet  known  to  warrant  any  definite  conclusions.  Here 
also  is  a  large  field  for  the  expert,  and  until  it  is  tilled  by  him  the 
results  can  hardly  be  expected  to  be  what  they  should  be.  As  I  have 
suggested,  the  best  results  may  here  be  expected  from  serum  treat- 
ment and  vaccine  treatment  conjointly. 


PNEUMOCOCCUS   INFECTIONS 

While  the  results  which  have  been  hitherto  obtained  in  the  serum 
treatment  of  pneumococcus  pneumonia  have  been  rather  disappoint- 
ing, Neufeld  and  Handel  have  recently  pointed  out  that  this  may 
have  been  due  in  part  to  the  administration  of  sera  of  too  low  a  titer 
and  of  insufficient  amounts,  in  part  to  the  administration  by  the 
subcutaneous  route,  and  in  part  to  the  use  of  sera  which  did  not 
correspond  to  the  same  strain  as  the  infecting  organism.  The  same 
observers  could  show  that  the  so-called  polyvalent  sera  of  commerce 
were  in  reality  not  truly  polyvalent,  and  possessed  protective  value 
only  against  a  certain  group  of  pneumococcus  strains,  while  they 
were  of  no  avail  against  other  types.  They  emphasize  that  no  cura- 
tive properties  can  be  expected  from  a  given  serum  unless  this  is 
homologous  for  the  type  that  is  causing  the  infection.  It  will  accord- 
ingly be  necessary  to  resume  the  serum  treatment  of  pneumonia 
from  this  standpoint,  and  to  establish  the  type  of  the  organism  in 
every  case  before  an  ultimate  verdict  upon  the  subject  can  be 
reached. 

A  serum  which  possesses  a  high  degree  of  protective  as  well  as 
curative  value  against  the  common  strains  of  pneumococci,  in  the 
animal  experiment,  is  at  present  prepared  under  Neufeld's  direction 
by  the  Serum  Institute  of  Saxony,  and  may  be  recommended  for 
use  in  infections  with  the  corresponding  strains.  Its  titer  is  such 
that  1  c.c.  will  protect  5000  grams  of  body  weight  (tested  against 
white  mice  weighing  from  18  to  20  grams)  against  0.1  to  0.0001  c.c. 
of  a  pneumococcus  bouillon  culture,  the  fatal  dose  being  0.000001 
or  less.  Neufeld  and  Handel  point  out  that  it  is  essential  to  test 
out  the  serum  against  large  or  medium  doses  of  the  organism  as  the 
values  obtained  in  the  case  of  small  doses  do  not  apply  to  correspond- 
ing multiples.  Of  a  serum,  for  example  which  would  protect  in  a 


PNEUMOCOCCUS  INFECTIONS  253 

dose  of  1  c.c.  against  the  minimal  fatal  dose,  5  c.c.  would  not  neces- 
sarily protect  against  five  multiples  of  the  fatal  dose  and  so  on.  They 
ascertained  that  there  is  a  certain  threshold  of  action  beyond  which 
a  given  dose  will  protect  against  many  multiples  (up  to  a  million) 
of  fatal  doses,  while  below  this  point  the  activity  of  the  serum 
rapidly  diminishes  and  may  indeed  be  nil. 

In  a  concrete  case  the  question  naturally  arises  whether  the  type 
of  the  infection  corresponds  to  the  strains  with  which  the  serum  was 
produced.  To  this  end  it  is  recommended  to  inject  a  mouse  with  a 
small  quantity  (0.5  c.c.)  of  the  patient's  sputum  to  which  one  titer 
dose  of  the  serum  has  been  added.  If  then  the  animal  does  not 
succumb  within  the  next  twenty-four  hours  we  may  assume  that  the 
serum  is  homologous  to  the  type  of  the  infection  and  would  hence  be 
applicable. 

Mode  of  Action. — As  regards  the  mode  of  action  of  his  antipneumo- 
coccus  serum  Neufeld  lays  special  stress  upon  its  bacteriotropic 
effect.  It  is  possible,  however,  that  it  may  have  a  certain  bacterio- 
lytic  as  well  as  antitoxic  component  as  well. 

Dosage  and  Mode  of  Administration. — As  the  absorption  from  the 
subcutaneous  tissue  is  notoriously  slow  the  serum  should  be  injected 
intravenously  whenever  possible,  or  intramuscularly,  if  for  any 
reason  (as  in  young  children)  the  other  route  is  excluded.  The  initial 
dose  for  an  adult  is  40  to  50  c.c.,  and  for  children  one-half  of  this 
quantity,  and  may  be  repeated  on  the  following  day.  If  the  patient 
has  been  previously  treated  with  horse  serum  it  is  recommended 
to  inject  a  small  quantity  (about  0.5  c.c.)  a  few  hours  before  the 
principal  injection  is  given,  the  idea  being  to  guard  against  anaphyl- 
actic  reactions  by  the  production .  of  the  antianaphylactic  state. 
Following  the  second  injection  no  anaphylactic  disturbances  need 
of  course  be  anticipated  (for  the  same  reason) .  Above  all  it  is  essen- 
tial to  inject  as  soon  as  the  diagnosis  has  been  made. 

Results. — Regarding  the  results  which  may  be  obtained  with 
antipneumococcic  sera  prepared  along  the  lines  which  have  been 
indicated  by  Neufeld  and  Handel,  it  would  be  premature  to  make  any 
definite  statements.  Suffice  it  to  say  that  the  treatment  has  no 
appreciable  influence  upon  the  pathological-anatomical  condition 
of  the  lung,  but  that  the  general  condition  of  the  patient  is  apparently 
improved  and  that  an  early  crisis  may  be  expected  if  the  serum  is 
administered  early.  To  what  extent  the  death-rate  will  be  affected 


254  PASSIVE  IMMUNIZATION 

by  the  treatment  is  impossible  to  foretell  from  the  meager  data  which 
are  now  available.  Of  the  thirty-seven  cases  treated  by  Betz  and 
Geronne,  five  died,  but  of  these  several  were  tubercular,  and  one 
in  an  extremely  critical  condition,  when  injected  (on  the  sixth  day). 

In  localized  infections  with  the  pneumococcus  it  would  seem  advis- 
able to  combine  the  serum  treatment  with  the  use  of  sodium  oleate 
and  boracic  acid,  as  recommended  by  Lamar  (see  section  on 
Chemotherapy) . 


STAPHYLOCOCCUS   INFECTIONS 

While  several  attempts  have  been  made  to  combat  staphylococcus 
infections  with  corresponding  antisera,  we  know  too  little  as  yet 
of  their  mode  of  action  and  their  effect  as  to  warrant  more  than  a 
mere  statement  of  this  fact.  Noteworthy  clinical  results  have  appar- 
ently not  as  yet  been  achieved.  The  great  question  here  as  else- 
where in  the  treatment  of  bacterial  infections  with  antisera  is 
whether  or  not  all  the  organisms  are  of  one  type.  If  this  should  not 
be  the  case  it  is  clear  that  only  a  homologous  serum  would  likely 
be  of  value,  and  as  a  matter  of  fact,  evidence  is  rapidly  accumulating 
which  goes  to  show  that  marked  differences  actually  exist  between 
staphylococci  derived  from  different  sources. 


ANTIGONOCOCCUS    SERUM 

Of  late  an  antigonococcus  serum  also  has  been  placed  upon  the 
market,  for  which  good  results  have  been  claimed.  Torrey's  serum 
is  prepared  by  immunizing  sheep  with  gradually  increasing  doses  of 
dead,  and  later,  of  living  cultures  of  virulent  strains,  and  is  marketed 
in  2  c.c.  ampoules,  which  amount  represents  a  single  dose.  Repeated 
injections  are  made  at  intervals  of  one,  two,  three,  or  four  days, 
according  to  the  requirements  of  the  individual  case.  Its  use  is 
advocated  in  chronic  conditions  produced  by  gonococcic  infection, 
as  in  those  arising  from  a  direct  extension  of  the  primary  infection 
into  organs  like  the  prostate,  epididymis,  testicles,  bladder,  and 
Fallopian  tubes,  as  also  in  cases  of  gonococcus  arthritis,  iritis,  endo- 


AUTOSERUM  THERAPY  255 

carditis,  pleuritis,  and  meningitis.  As  yet  not  enough  is  known  of 
the  effect  of  the  injection  upon  the  maladies  in  question  to  warrant 
any  definite  statements. 

In  the  booklet  on  the  subject  which  has  been  issued  by  the  manu- 
facturers the  statement  is  made  that  within  a  year  10,000  doses  of 
the  serum  had  been  sent  out  for  experimental  purposes,  and  that  of 
the  cases  reported  upon  58  per  cent,  showed  decided  benefit,  and  that 
in  17  per  cent,  only  the  results  had  not  been  favorable.  Future 
investigations  here  also  are  needed  to  establish  the  actual  status  of 
the  treatment,  which,  a  priori,  of  course,  would  seem  logical,  and 
expecially  so  when  combined  with  corresponding  vaccination. 


AUTOSERUM   THERAPY 

A  number  of  investigators  have  suggested  the  administration 
of  the  patient's  own  serum  under  various  pathological  conditions 
upon  grounds,  it  must  be  admitted,  which  in  some  cases  seem  rather 
lacking  in  force.  Gilbert  and  Fede  thus  recommend  the  subcutaneous 
injection  of  small  quantities  (1  to  2  c.c.)  of  the  patient's  own  exudate 
in  the  treatment  of  tubercular  peritonitis  and  pleurisy,  and  claim 
that  the  absorption  of  the  exudate  is  thus  greatly  hastened.  Similar 
results  have  been  reported  by  Senator  and  Schniitgen.  Modinos 
reports  that  the  injection  of  moderate  doses  (about  8  c.c.)  of  the 
patient's  own  serum  is  of  beneficial  influence  upon  the  course  of 
various  acute  infectious  diseases,  and  cites  specific  cases  of  influenza, 
typhoid,  and  Malta  fever.  Others  have  reported  favorable  results 
from  the  injection  of  small  doses  (1  to  2  c.c.)  of  serum  in  gonorrheal 
arthritis,  and  so  on. 

A  few  years  ago  Hodenpyle  met  with  an  instance  of  carcinoma- 
tosis  associated  with  chylous  ascites  in  which  the  disease  seemed 
to  have  been  arrested,  and  in  wrhich  he  assumed  that  protective 
antibodies  (cytotoxins)  were  present  in  the  circulation.  He  used 
this  fluid  in  large  quantities  in  a  number  of  cases  of  cancer,  and  for 
a  time  apparently  with  a  beneficial  effect.  The  results,  however, 
were  not  lasting,  and  so  far  as  my  knowledge  goes  none  of  the  treated 
patients  are  now  living.  The  donor,  moreover,  died  within  a  year 
of  the  time  the  treatment  of  the  patients  was  begun.  Following 
these  experiments  a  number  of  investigators  used  the  ascitic  fluid 


256  PASSIVE  IMMUNIZATION 

of  cancer  cases  in  the  treatment  of  the  corresponding  patients,  but 
likewise  without  any  lasting  benefit. 

Autoserum  Therapy  in  Syphilis  of  the  Central  Nervous  System. — 
Of  late  the  patient's  own  serum  has  been  advocated  in  the  treat- 
ment of  syphilitic  diseases  of  the  central  nervous  system,  the  serum 
being  administered  by  the  subdural  route,  and  obtained  from  the 
patient  one  hour  after  an  intravenous  injection  of  salvarsan.  The 
underlying  idea  is  to  bring  both  the  salvarsan  and  any  antibodies 
that  may  be  formed  in  the  patient's  own  body  into  as  intimate  a 
contact  as  possible,  with  the  spirochetes,  located  in  the  perivascular 
lymph  spaces  and  the  adventitia  of  the  bloodvessels.  This  plan 
seems  thoroughly  logical  and  deserves  extended  investigation. 
That  protective  antibodies  are  actually  present  in  the  blood  serum 
of  treated  syphilitic  patients  has  been  demonstrated  beyond  a 
reasonable  doubt.  It  has  thus  been  observed  by  Taege  that  the  milk 
of  syphilitic  mothers,  after  treatment  with  salvarsan,  develops 
curative  properties  for  the  infected  infants,  and  other  investigators 
have  noted  marked  improvement  in  adults  following  the  injection 
of  the  serum  of  treated  syphilitic  individuals.  The  effects,  however, 
were  not  lasting,  and  it  hence  appears  rational  to  combine  the  use 
of  the  salvarsan  with  that  of  the  serum.  The  salvarsan,  moreover, 
when  given  by  itself,  by  the  subdural  route  is  too  irritating  to  warrant 
its  use  in  this  manner,  while  in  combination  with  serum  this  objec- 
tionable effect  apparently  falls  away. 

Technique. — The  procedure  which  has  been  advocated  by  Swift 
and  Ellis  is  the  following:  One  hour  after  an  injection  of  salvarsan 
or  neosalvarsan,  50  c.c.  of  blood  are  withdrawn  from  one  of  the  veins 
at  the  bend  of  the  elbow,  to  which  end  a  MacRae  puncture  needle 
will  be  found  most  convenient,  the  blood  being  aspirated  directly 
into  large  tubes.  The  serum  is  allowed  to  separate  out  overnight 
(in  the  ice-box),  and  the  following  morning  is  diluted  to  40  per 
cent,  with  normal  saline.  At  that  time  approximately  15  c.c.  of 
spinal  fluid  are  withdrawn  and  replaced  by  30  c.c.  of  the  diluted 
serum,  which  has  previously  been  heated  for  one-half  hour  at  56°  C. 
and  then  brought  to  the  temperature  of  the  body.  The  patients  are 
kept  in  bed  for  twenty-four  hours,  the  foot  end  being  raised  for 
about  one  hour  following  the  treatment. 

After  two  or  three  weeks  the  injection  is  repeated  and  so  on, 
according  to  the  symptoms  of  the  individual  case.  As  a  rule  there 


NORMAL  SERUM  THERAPY  257 

is  but  little  reaction  after  the  injection,  except  in  tabetics  in  whom 
lightning  pains  may  occur,  or  become  more  violent  for  a  while,  if 
they  previously  existed. 

Results. — Too  little  time  has  elapsed  since  this  method  of  treat- 
ment was  first  advocated,  and  too  small  a  number  of  patients  has 
as  yet  been  treated  to  warrant  any  far-going  conclusions. 

Swift  and  Ellis  mention  four  tabetics,  in  whom  the  cell  count  in 
the  cerebrospinal  fluid  promptly  fell  to  normal,  while  the  globulins 
decreased  in  amount  much  more  rapidly  than  during  the  previous 
treatment  with  salvarsan  and  mercury  alone;  at  the  same  time  the 
Wassermann  reaction  in  the  spinal  fluid  became  negative  in  two 
of  the  patients,  even  when  0.5  c.c.  of  fluid  was  used  in  the  test. 
In  two  other  patients,  however,  the  treatment  had  little  effect  on 
the  Wassermann. 

As  the  writers  suggest,  future  experience  may  show  that  the  best 
results  may  be  obtained  by  further  enforcing  the  beneficial  effect 
of  the  serum  by  the  addition  of  neosalvarsan.  They  mention  that 
in  one  instance  the  patient  received  0.5  milligram  of  the  neosalvarsan 
diluted  with  12  c.c.  of  normal  serum  plus  18  c.c.  of  normal  saline, 
as  a  first  injection,  which  was  followed  ten  days  later  by  one  of 
1  milligram,  similarly  diluted,  without  causing  any  undue  reaction. 


NORMAL    SERUM   THERAPY 

Under  various  pathological  conditions  normal  serum  also  has  been 
injected  for  curative  purposes  and  favorable  results  have  been 
observed  in  many  instances.  A  number  of  investigators  thus  report 
a  remarkable  influence  upon  certain  toxicoses  of  pregnancy.  Mayer 
mentions  several  cases  of  various  types  of  dermatoses  (herpes, 
urticaria,  pruritus)  besides  a  case  of  eclampsia,  and  one  of  acro- 
paresthesia  of  the  finger  tips,  in  which  excellent  results  followed 
the  injection  of  serum  from  a  normal  case  of  pregnancy.  He  con- 
cluded that  the  serum  of  the  patients  in  question  was  deficient 
in  normal  protective  substances  against  the  poisons  which  are 
absorbed  from  the  developing  embryo,  and  that  these  substances 
are  specific  in  their  nature.  Freund,  on  the  other  hand,  while  con- 
firming the  beneficial  effects  of  the  serum  of  normally  pregnant 
women  upon  the  toxicoses  referred  to,  obtained  equally  satisfactory 
17 


258  PASSIVE  IMMUNIZATION 

results  with  normal  serum  from  non-pregnant  women,  as  also  from 
men,  horses,  etc.  Corresponding  observations  have  since  been 
published  by  others. 

Favorable  results  have  also  been  observed  by  many  investigators, 
following  the  injection  of  normal  serum  or  of  defibrinated  blood, 
in  various  hemorrhagic  conditions.  Weil  thus  found  that  the  subcu- 
taneous injection  of  30  c.c.,  or  the  intravenous  injection  of  15  c.c.  of 
fresh  human  or  animal  serum  has  a  markedly  beneficial  effect  upon 
the  bleeding  of  hemophiliacs.  Leary  reports  several  cases  in  which 
following  a  preliminary  injection  of  30  c.c.  of  normal  rabbit  serum 
operations  could  be  carried  on  without  subsequent  bleeding  of  note. 
Welch  cites  twelve  cases  of  hemophilia  neonatorum,  all  of  which 
recovered  after  the  subcutaneous  injection  of  fresh  human  serum 
(average,  80  c.c.  in  10  c.c.  d6ses,  distributed  over  four  days). 

Other  observers  obtained  favorable  results  in  cases  of  rheumatic 
purpura  with  intestinal  hemorrhages,  in  uterine  bleeding,  in  intes- 
tinal bleeding  in  connection  with  cirrhosis  of  the  liver  and  typhoid 
fever,  in  hemorrhagic  retinitis,  etc.  Moss  suggests  that  in  those  cases 
in  which  a  notable  degree  of  anemia  has  already  developed,  the  in- 
jection of  large  quantities  of  defibrinated  human  blood  is  probably 
the  best  procedure,  care  being  taken  that  a  donor  is  selected  whose 
serum  will  not  agglutinate  or  hemolyze  the  red  corpuscles  of  the 
recipient.  In  other  cases  normal  rabbit  serum  will  be  found  to  be 
just  as  efficacious.  It  has  the  advantage,  moreover,  of  being  obtain- 
able without  difficulty,  of  being  very  little  toxic,  while  the  chances 
that  the  patient  has  been  rendered  hypersensitive  by  previous  injec- 
tions of  rabbit  serum  are  rather  remote. 

While  human  blood  has  also  been  used  quite  extensively  in  the 
treatment  of  advanced  cases  of  pernicious  anemia,  leukemia,  splenic 
anemia  and  pseudoleukemia,  lasting  effects  have  not  been  achieved, 
while  the  immediate  results,  owing  no  doubt  to  the  introduction 
of  the  large  number  of  normal  red  cells,  may  be  quite  satisfactory. 


CHAPTER  XIV 
CHEMOTHERAPY 

IN  the  foregoing  chapters  we  have  seen  that  the  animal  body 
has  at  its  disposal  a  mechanism  by  means  of  which  it  is  not  only 
capable  in  many  instances  of  preventing  infection,  but  even  of  over- 
coming this  successfully  if  by  any  chance  microorganisms  have 
once  passed  the  outer  barriers  and  have  gained  a  foothold  in  the 
tissues  proper.  We  have  also  seen  that  it  is  possible  to  introduce 
some  of  those  substances  which  the  body  makes  use  of  in  its  defence, 
from  without,  and  that  we  can  frequently  turn  the  balance  of  the 
scales  toward  recovery  in  this  manner,  where  unaided  this  would 
have  been  impossible,  or  attended  by  grave  danger.  Nevertheless 
we  must  admit  that  only  too  often  all  our  efforts  to  combat  infection 
by  the  body's  own  methods  are  in  vain,  and  that  in  the  majority  of 
infections  we  are  still  far  from  a  successful  treatment. 

In  view  of  the  fact  that  in  the  test-tube  we  are  able  to  destroy 
microorganisms  with  the  greatest  ease,  by  the  aid  of  a  large  number 
of  chemical  preparations,  the  thought  has  naturally  suggested  itself 
whether  it  would  not  be  possible  to  assist  the  normal  defences  of 
the  body  by  the  administration  of  some  of  these  substances.  We 
know  as  a  matter  of  fact  that  the  only  specific  medicinal  treatment 
of  the  older  Pharmacopcea,  viz.,  that  of  malaria  by  means  of  quinine, 
and  of  syphilis  by  means  of  mercury,  depends  upon  the  destructive 
effect  of  the  remedies  in  question  upon  the  respective  parasites. 
The  recognition  of  this  fact  is  of  recent  date,  however,  and  does 
not  form  the  basis  upon  which  the  treatment  of  these  diseases  was 
established.  The  discovery  of  the  therapeutic  properties  of  quinine 
and  mercury,  in  other  words,  was  not  the  outcome  of  logical  thought 
and  corresponding  experimentation,  but  purely  accidental. 

But  the  fact  that  it  is  actually  possible  to  destroy  some  of  the 
pathogenic  microorganisms  in  the  body  of  an  infected  individual  by 
chemical  means,  would  suggest  that  a  similarly  fortunate  result 
might  be  achieved  with  other  substances  in  the  case  of  other  organ- 


260  CHEMOTHERAPY 

isms.  The  earlier  investigations  in  this  direction  were,  however,  not 
crowned  with  success,  and  it  was  soon  realized  that  in  these  studies 
also,  accident  would  probably  have  to  play  a  role,  unless  indeed 
every  chemical  substance  were  individually  tested.  The  first  and 
most  formidable  difficulty  which  was  encountered  depended  upon 
the  fact  that  the  majority  of  those  substances  which  have  strong 
germicidal  properties,  when  tested  outside  of  the  animal  body,  were 
promptly  rendered  innocuous  by  entering  into  chemical  combination 
with  the  albumins  of  the  blood,  when  introduced  into  the  body, 
and  if  a  certain  dose  was  exceeded  their  toxic  effect  was  such  that 
any  attempt  at  destruction  of  the  parasites  would  have  carried 
with  it  the  destruction  of  the  host.  I  well  recall  an  interesting 
observation  which  illustrates  this  point.  A  patient  suffering  from 
pneumonia  was  accidentally  given  a  dose  of  bichloride  of  mercury 
which  nearly  caused  the  individual's  death.  He  was  saved  with 
difficulty,  but  died  of  his  pneumonia  a  few  days  later.  Evidently 
the  dose  of  the  bichloride,  though  large  enough  to  have  nearly 
killed  the  patient,  had  not  been  sufficiently  large  to  kill  the  offending 
parasites. 

Organotropism  and  Parasitotropism. — In  work  of  this  nature  the  dis- 
tribution of  the  poison  in  the  body  is  evidently  of  prime  importance. 
If,  aside  from  any  binding  action  on  the  part  of  the  circulating 
albumins,  the  affinity  of  the  poison  is  greater  for  the  tissues  of  the 
body  than  for  the  protoplasm  of  the  parasites,  or  to  use  the  parlance 
of  Ehrlich,  if  the  poison  is  more  markedly  organotropic  than  parasito- 
tropic,  it  is  evidently  not  suitable  for  therapeutic  purposes,  and 
especially  so,  if  at  the  same  time  the  toxic  dose  for  the  macroorgan- 
ism  should  be  smaller  than  that  for  the  microorganism.  The  great 
problem  then  has  been  to  discover  substances  which,  while  possessed 
of  germicidal  properties,  or  what  amounts  to  the  same  thing,  of  the 
power  to  inhibit  reproduction  of  the  parasites,  shall  also  be  non- 
toxic,  or  but  little  toxic  for  the  macroorganism,  and  more  markedly 
parasitotropic  than  organotropic. 

In  this  investigation,  Ehrlich,  to  whom  we  are  already  indebted 
for  so  much  of  our  knowledge  of  the  more  intricate  problems  of  cell 
life,  has  again  taken  the  leading  position,  and  may  indeed  very 
appropriately  be  styled  the  father  of  modern  pharmacology  and 
chemotherapy.  Since  the  degree  of  antibody  formation  in  systemic 
infections  with  protozoan  parasites,  in  contradistinction  to  bacterial 


CHEMORECEPTORS  261 

infections,  is  usually  insufficient  in  itself  to  successfully  combat 
the  corresponding  maladies,  the  attention  of  Ehrlich  and  his  col- 
laborators, and  many  other  noted  investigators,  has  within  recent 
years  been  largely  centred  upon  these  very  infections.  As  a  result 
of  the  study  of  several  thousand  different  products  in  regard  to 
their  influence  upon  trypanosomes  which  are  especially  convenient 
test  objects  in  this  respect,  it  has  been  found  that  there  are  after  all 
very  few  which  can  effect  a  cure  in  animals  that  have  been  infected 
with  the  parasite  in  question,  but  these  are  well  characterized  chemi- 
cally, and  belong  to  three  distinct  groups.  The  first  of  these  com- 
prises certain  arsenical  preparations,  notably  arsenious  acid,  atoxyl 
(arsanil),  arsacetin,  arsenophenyl  glycin,  and  the  dichlorhydrate  of 
dioxydiaminoarsenobenzol  (popularly  known  as  preparation  No. 
606),  and  in  addition  to  these  certain  antimony  preparations.  The 
second  group  is  represented  by  certain  azo  dyes,  such  as  trypan-red, 
trypan-blue,  and  trypan-violet,  while  certain  basic  triphenylmethane 
dyes,  such  as  parafuchsin,  methyl  violet,  pyronin,  and  others,  belong 
to  the  third  order. 

Chemoreceptors. — The  study  of  these  products  in  their  behavior 
to  trypanosomes  has  led  to  a  number  of  interesting  discoveries. 
While  Ehrlich  originally  assumed  that  the  so-called  side  chains  of 
the  protoplasmic  molecule  only  served  purposes  of  nutrition,  in 
other  words,  that  all  receptors  were  essentially  nutriceptors,  and  that 
medicinal  agents  were  not  bound  in  this  manner,  he  now  holds  that 
receptors  do  exist  by  which  such  substances  may  be  bound,  and 
terms  these  chemoreceptors. 

He  suggests  that  the  groups  of  the  latter  order  in  accordance  with 
their  simpler  functions  are  probably  of  a  less  complex  structure, 
that  they  are  more  firmly  attached  to  the  cell,  and  are  hence  less 
readily  cast  off,  and  that  as  a  consequence  of  their  "sessile"  character, 
crystalline  chemical  substances  are,  generally  speaking,  incapable  of 
eliciting  the  liberation  of  corresponding  antibodies.  This  conclusion 
was  based  upon  the  following  observation: 

If  a  given  strain  of  trypanosomes  is  continuously  treated  with 
chemotherapeutic  agents  belonging  to  one  of  the  groups  referred  to 
above,  a  race  of  organisms  develops  which  can  no  longer  be  influenced 
by  that  particular  product  and  which  is  accordingly  said  to  be  "fast/' 
in  reference  to  that  particular  drug.  It  is  interesting  to  note  that 
this  acquired  resistance  or  "fastness"  is  in  a  large  measure  specific. 


262  CHEMOTHERAPY 

A  strain  which  has  been  rendered  resistant  to  trypan-red  and 
which  is  also  fast  to  trypan-blue  and  violet  is  thus  non-resistant 
to  arsenic  and  the  triphenylmethane  dyes,  while  one  which  has 
been  rendered  arsenic-fast  is  resistant  only  to  this  and  not  to  the 
trypan-dyes  and  the  triphenylmethanes,  etc.  This  fastness,  it  was 
then  ascertained,  remained  a  constant  character  through  innumerable 
generations,  so  long  in  fact  as  the  organism  multiplies  by  direct 
division,  while  it  is  lost  in  the  descendants  of  sexual  reproduction. 

The  attempt  to  explain  the  development  of  such  drug-fast  strains, 
as  I  have  just  said,  led  Ehrlich  to  assume  the  existence  of  chemo- 
receptors.  Since  arsanil  itself  is  non-toxic,  while  its  reduction  prod- 
ucts are  capable  of  killing  trypanosomes  in  high  dilution,  it  follows 
that  the  trivalent  arsenical  radicle  which  is  in  combination  with  the 
benzoyl  radicle  must  in  some  manner  be  anchored  to  the  trypano- 
somes; and  as  the  toxic  effect  of  the  arsanil  is  lost  in  the  so-called 
arsenic  fast  strains,  the  conclusion  suggests  itself  that  the  untreated 
parasite  must  possess  a  definite  group  or  receptor  with  which  the 
arsenical  group  can  unite,  and  which  is  capable  of  undergoing  a 
certain  modification,  in  consequence  of  which  its  affinity  for  the 
preparation  in  question  is  lost,  or  at  least  diminished.  In  the  absence 
of  such  a  combining  group  it  would  be  very  difficult  to  explain  why 
the  treated  strain  should  be  arsenic  resistant  and  the  non-treated 
arsenic  susceptible. 

Drug  "Fastness." — In  a  former  chapter  we  have  seen  that  a  certain 
type  of  immunity  results  from  the  occurrence  of  receptoric  atrophy,' 
and  Ehrlich  has  shown  that  during  the  process  of  serum  immuniza- 
tion, trypanosomes  may  develop  a  serum  fastness  which  is  of  this 
character;  where,  in  other  words,  those  nutriceptors  of  the  parasite, 
which  are  continuously  occupied  by  a  corresponding  antibody  fur- 
nished by  the  host  (the  rat  for  example),  disappear  or  are  replaced 
by  receptors  of  a  different  structure,  through  which  the  nutrition  of 
the  cell  can  again  be  carried  on.  In  studying  the  nature  of  drug  fast- 
ness, Ehrlich  then  ascertained  that  this  is  not  dependent  on  atrophy 
of  the  corresponding  receptors,  but  upon  a  modification  in  their 
structure,  as  is  evidenced  by  the  fact  that  by  changing  the  structure 
of  the  arsenical  product,  for  example,  this  may  still  be  forced  upon 
the  parasite,  so  to  speak,  and  lead  to  its  destruction. 

The  recognition  of  this  possibility  is,  of  course,  of  the  greatest 
importance,  as  it  shows  the  lines  along  which  such  parasiticidal 


DRUG  FASTNESS  263 

substances  must  be  constructed,  in  order  to  produce  the  maximum 
amount  of  effect  with  the  least  chance  of  leading  to  the  development 
of  an  insurmountable  drug  resistance.  That  this  can  be  done, 
Ehrlich  himself  has  demonstrated  in  a  perfectly  satisfactory  manner. 
He  could  thus  show  that  mice  which  had  been  infected  with  arsanil- 
fast  trypanosomes  could  be  cured  with  arsenophenyl  glycin,  even  at 
a  time  when  death  seemed  to  be  imminent.  The  problem  will,  of 
course,  be  the  more  difficult  the  larger  the  number  of  drug-fast 
strains  that  is  possible,  and  not  only  this,  but  the  larger  the  number 
of  serum-fast  strains  that  may  develop.  For  we  must  bear  in  mind 
that  the  destruction  of  the  parasites  in  question  is  of  necessity 
followed  by  the  development  of  corresponding  antibodies,  which  in 
itself  is,  of  course,  a  favorable  occurrence. 

If,  however,  the  destruction  of  the  trypanosomes  by  the  arsenical 
preparation,  for  example,  has  not  been  complete,  and  if  the  resultant 
antibodies  do  not  succeed  in  killing  off  the  rest,  there  is  a  strong 
probability  that  a  serum-fast  strain  will  now  develop  and  bring  about 
a  relapse  (relapse  strain  No.  I).  When  some  of  these  organisms 
then  die  or  are  killed  by  a  repetition  of  the  dose  of  arsenic,  if  indeed 
the  strain  is  still  susceptible  to  the  same  preparation,  a  new  type 
of  antibody  will  be  formed  which  will  be  specifically  directed  against 
relapse  strain  No.  I,  from  which  a  new  serum-fast  strain  may  then 
develop  and  cause  a  second  relapse  (relapse  strain  No.  II),  and  so 
on,  the  number  of  serum-fast  strains  being  limited  only  by  the  ability 
of  the  parasite  to  produce  new  receptors  with  which  it  can  attend 
to  its  nutrition. 

This  implies,  of  course,  that  as  the  number  of  different  kinds  of 
foodstuffs  which  the  parasite  can  utilize,  progressively  diminishes, 
a  time  will  finally  come  when  the  infection  will  become  eradicated 
spontaneously.  This  might  occur  relatively  early,  so  that  the 
infected  animal  or  patient  would  actually  receive  the  benefit  of  this 
fractional  destruction  of  the  parasites,  but,  on  the  other  hand,  there 
is  a  possibility  that  during  each  relapse  vital  structures  may  be 
damaged  to  such  an  extent  that  the  individual  would  not  live  long 
enough  to  reach  the  point  where  the  infection  would  at  last  have 
exhausted  itself. 

Examples  in  point  are  relapsing  fever,  on  the  one  hand,  and  syphilis 
and  sleeping  sickness,  and  possibly  also  malaria,  on  the  other.  In 
relapsing  fever  we  thus  have  evidence  that  only  three  or  four  different 


264  CHEMOTHERAPY 

serum-fast  strains  can  exist,  and  we  accordingly  find  that  after  a 
patient  has  safely  passed  through  the  two  or  three  corresponding 
relapses,  spontaneous  recovery  occurs,  there  being  then  antibodies 
present  against  the  only  strains  that  are  possible  in  that  particular 
milieu.  In  syphilis  it  is  quite  different.  Here  the  number  of  food- 
stuffs that  the  spirochete  can  utilize  is  evidently  quite  large.  In  the 
untreated  individual,  relapse  follows  relapse,  and  the  damage  done 
to  vital  parts  is  only  too  often  so  extensive,  relatively  early  in  the 
course  of  the  infection,  that  the  patient  succumbs,  owing  to  the 
resultant  injury,  long  before  the  disease  has  "worn  itself  out." 

The  discovery  of  this  element  of  "fastness"  or  resistance  on  the 
part  of  microorganisms  is  evidently  of  the  greatest  importance,  as 
it  throws  light  upon  many  phenomena,  the  cause  of  which  has  here- 
tofore been  most  obscure.  The  question  has  thus  long  remained 
unanswered,  why  the  syphilitic  individual  cannot  be  re-inoculated 
with  syphilitic  virus  while  his  disease  is  active.  The  reason  now  is 
quite  evident.  For  we  know  that  the  spirochetal  strains  which  at 
any  time  are  operative  in  the  body  of  the  syphilitic  patient  are 
"fast"  strains,  of  varying  degree,  and  that  antibodies  are  present 
in  his  blood  which  are  specifically  "tuned"  to  those  of  a  lower 
order,  i.  e.,  to  those  serum  strains  from  which  the  "highest"  ones 
have  become  developed,  so  that  the  "street  spirochete,"  so  to  speak, 
if  introduced  under  such  conditions,  must  of  necessity  meet  with 
those  antibodies  which  would  lead  to  their  destruction.  If  once  it 
were  possible  to  cultivate  all  these  different  strains,  then  it  would 
also  be  possible  to  reinfect  the  syphilitic  individual,  not  with  the 
street  virus  to  be  sure,  but  with  a  strain  of  a  higher  order  of  serum 
fastness,  than  would  correspond  to  the  number  of  antibodies  that 
have  already  been  formed.  In  the  animal  experiment,  using  try- 
panosomes,  this  can  indeed  be  done  at  the  present  time,  and  at  the 
Speyer  Haus,  in  Frankfurt,  Ehrlich  has  under  cultivation  all  five  of 
the  serum-fast  strains  which  are  possible  in  the  organism  of  the  mouse. 

Therapia  Magna  Sterilisans. — As  the  development  of  "fast"  strains 
is  thus  one  of  the  greatest  impediments  to  the  successful  treatment 
of  the  maladies  in  question,  our  efforts  should  be  directed  to  the 
discovery  of  medicinal  substances  which  should  be  capable  of  effect- 
ing the  complete  sterilization  of  the  individual  at  one  time  (Ehrlich's 
therapia  magna  sterilisans) .  The  demonstration  that  this  is  actually 
possible,  not  only  in  the  infected  animal,  but  also  in  the  infected 
human  being,  we  also  owe  to  the  indefatigable  genius  of  Ehrlich. 


Showing  the  Effect  of  Salvarsan  Treatment  upon  Spirillosis 
of  Chickens.    (Taken  from  Ehrlieh  and  Hata.) 

A,  four  days  after  infection,  untreated;  B,  two  days  after  infection, 
treated;  C,  control  showing  spirilla  ;  D,  a  treated  chicken  showing  absence 
of  spirilla. 


SALVARSAN  AND  ITS  USE  IN  TREATMENT  OF  SYPHILIS     265 

To  give  an  idea  of  the  immense  amount  of  labor  which  this  work 
has  involved  it  will  be  sufficient  to  point  out  that  up  to  the  year 
1910  over  six  hundred  arsenical  products  alone  had  been  prepared 
and  tested  biologically  and  therapeutically  under  Ehrlich's  direction. 
Of  these  the  one  carrying  the  number  606  has  been  of  special  interest 
to  clinicians,  as  its  wonderful  therapeutic  effect  upon  trypanosome 
infections  and  certain  spirilloses  of  animals  (notably  the  spirillosis 
of  relapsing  fever  and  chicken  spirillosis)  suggested  the  idea  that  the 
product  might  be  similarly  effective  in  the  treatment  of  human 
syphilis  (see  Plate  IV).  After  preliminary  studies  had  then  shown 
that  a  single  dose  of  the  substance  is  capable  of  causing  the  complete 
destruction  of  all  spirochetes  in  syphilis-infected  rabbits,  with  the 
complete  cure  of  the  testicular  chancre  and  without  the  occurrence 
of  a  relapse,  it  was  clearly  indicated  that  corresponding  experiments 
in  the  human  being  were  justifiable  (see  Plate  V).  After  the  first 
trials  in  this  direction  had  then  demonstrated  a  similarly  beneficial 
effect,  Ehrlich  placed  the  remedy  in  the  hands  of  a  large  number 
of  special  workers  in  this  field  in  order  that  a  conclusion  should  be 
reached  as  soon  as  possible  regarding  its  therapeutic  value,  the 
indications  and  contra-indications  to  its  use,  the  question  of  dosage, 
mode  of  administration,  etc.  As  a  consequence  reports  on  these 
questions  could  be  collected  within  a  year,  covering  the  administration 
of  the  remedy  in  many  thousands  of  cases,  so  that  in  a  relatively 
short  time  the  verdict  could  be  reached  that  preparation  606,  or 
salvarsan,  as  it  is  now  termed,  actually  constitutes  the  most  potent 
remedy  which  we  have  at  our  disposal  for  the  treatment  of  syphilis; 
and  we  would  emphasize  once  more  that  this  discovery  was  not  the 
result  of  accident,  but  the  outcome  of  carefully  planned  experimentation, 
carried  to  its  logical  issue. 


SALVARSAN  AND  ITS  USE  IN  THE  TREATMENT  OF   SYPHILIS 

Chemically  speaking  salvarsan  (Ehrlich's  "606")  is  the  dichlor- 
hydrate  of  dioxydiaminoarsenobenzol : 


As         =         As 
/\ 


NH 


2  I  J 

OH  OH 


NH 


266  CHEMOTHERAPY 

It  is  a  fine  yellow  powder,  easily  soluble  in  water,  methyl  alcohol, 
and  glycerin,  less  easily  soluble  in  ethyl  alcohol  and  insoluble  in 
ether.  Owing  to  the  readiness  with  which  it  undergoes  oxidation 
and  gives  rise  to  highly  poisonous  products,  it  is  marketed  in  little 
ampoules  from  which  the  air  has  been  removed  and  replaced  with 
an  indifferent  gas. 

Method  of  Application. — While  the  substance  was  originally  in- 
jected in  acid  solution,  i.  e.,  merely  dissolved  in  water,  this  method 
was  found  inapplicable  owing  to  the  intense  pain  which  followed 
its  use,  and  at  present  it  is  employed  practically  only  in  alkaline 
solution,  which  is  administered  intravenously,  and  should  be  freshly 
prepared  just  before  the  injection.  This  is  done  in  a  sterile  bottle 
of  about  500  c.c.  capacity,  graduated  in  50  c.c.,  and  containing 
some  large  sterile  glass  beads.  The  salt  solution  (0.65  per  cent.) 
which  is  used  as  solvent  and  diluent  should  be  freshly  prepared  from 
freshly  distilled  water  and  chemically  pure  sodium  chloride.  30  to 
40  c.c.  of  this  are  placed  in  the  bottle  and  the  dose  of  salvarsan 
added,  which  then  dissolves  on  vigorous  shaking. 

To  obtain  the  alkaline  solution  0.19  c.c.  of  a  15  per  cent,  solution 
of  caustic  soda  (NaOH)  are  now  added  for  every  0.1  gram  of  the 
remedy,  the  immediate  effect  being  the  formation  of  a  precipitate 
which  dissolves  on  shaking  and  then  gives  rise  to  a  clear  golden 
yellow  solution.  This  is  finally  diluted  with  the  sterile  saline  (warmed 
to  body  temperature),  such  that  every  50  c.c.  shall  correspond  to  0.1 
gram  of  salvarsan.  Taking  an  adult  dose  of  0.6  gram  as  example, 
the  final  bulk  would  thus  be  300  c.c.  Should  the  solution  not  be 
absolutely  clear  a  few  additional  drops  of  the  NaOH  solution  may 
be  added.  Occasionally  the  fluid  does  not  clear  up  upon  the  further 
addition  of  alkali,  in  which  event  it  is  probably  best  to  break  a 
new  ampoule  of  the  drug. 

To  give  the  injection,  an  ordinary  infusion  bottle  or  similar  con- 
trivance (properly  sterilized,  of  course)  is  arranged  at  the  bedside 
of  the  patient  and  charged  with  a  small  quantity  of  warmed  salt 
solution  which  should  completely  fill  the  rubber  tube  leading  to  the 
needle,  as  well  as  the  lumen  of  the  latter.  This  need  not  be  of  large 
caliber;  a  No.  18  (B.  &  S.  standard)  is  quite  sufficient  in  size.  The 
arm  having  been  cleansed,  at  the  bend  of  the  elbow,  with  soap 
and  water,  bichloride,  alcohol,  and  ether,  or,  has  recently  been  advo- 
cated, merely  painted  with  tincture  of  iodine,  about  the  site  of  the 


SALVARSAN  AND  ITS  USE  IN  TREATMENT  OF  SYPHILIS     267 

injection,  the  needle  is  plunged  into  any  one  of  the  large  veins  which 
there  present  themselves,  and  which  have  been  rendered  prominent 
by  constricting  the  upper  arm  with  a  bandage,  or  a  piece  of  rubber 
tubing,  without,  however,  obliterating  the  arterial  flow.  The  tourni- 
quet is  then  removed,  the  clamp  opened  on  the  rubber  tube,  and  the 
saline  allowed  to  flow.  If  the  result  shows  that  the  needle  is  actually 
in  the  vein,  the  salvarsan  solution  is  added  to  the  small  amount  of 
saline  remaining  in  the  bottle,  and  the  infusion  allowed  to  proceed. 
In  the  end  about  50  c.c.  of  saline  are  allowed  to  follow  the  salvarsan, 
so  that  the  tissue  about  the  site  of  the  puncture  shall  be  irritated 
as  little  as  possible  in  the  event  of  a  little  leakage  while  the  needle 
is  being  withdrawn. 

A  very  convenient  apparatus  for  the  administration  of  salvarsan 
is  that  suggested  by  Gary  (Fig.  16). 

FIG.  16 


Syringe  for  injection  of  salvarsan.     Glass  handle  guides  operator.     Two  bottles  enable 
alternate  use  of  drug  or  water. 

Special  care  should  be  had  that  the  injection  is  made  slowly,  and 
under  no  circumstances  should  it  be  allowed  to  consume  less  time 
than  twelve  to  fifteen  minutes.  Want  of  attention  to  this  point 
may  result  in  the  development  of  serious  symptoms.  While  it  is 
usual  that  the  patient's  face  becomes  flushed  during  the  injection, 
the  infusion  should  be  stopped  at  once,  if  sudden  pallor  develops, 
or  the  pulse  becomes  weak. 

Following  the  injection  the  patient  should  be  placed  in  bed  and 
should  remain  there  for  twenty-four  hours  or  longer,  according  to 
the  symptoms  which  develop  during  this  time.  To  give  the  injec- 
tion in  the  physician's  office,  and  then  to  allow  the  patient  to  go 
home  and  follow  his  usual  occupation,  is  a  dangerous  practice, 


268  CHEMOTHERAPY 

unless  indeed  the  dose  be  small,  and  the  quantity  of  fluid  less  than 
150  c.c. 

Other  methods  of  administering  salvarsan  have  practically  been 
abandoned,  and  we  would  indeed  warn  the  practitioner  against  the 
use  of  subcutaneous  and  intramuscular  injections,  no  matter  in 
what  medium  the  drug  may  have  been  dissolved  or  suspended. 
Such  injections  usually  cause  a  great  deal  of  pain  and  are  not 
infrequently  followed  by  necrosis. 

Reaction. — The  reaction  which  follows  the  intravenous  adminis- 
tration of  salvarsan  is  essentially  of  the  same  character  as  that 
following  the  injection  of  a  corresponding  amount  of  saline,  and 
varies  in  intensity  with  the  individual  case.  In  many  instances 
the  patient  is  not  inconvenienced  in  the  least.  At  first  when  it  was 
not  known,  that  it  is  essential  to  use  freshly  distilled,  sterile  water 
in  making  up  the  solution,  it  was  only  too  common  to  meet  with 
fairly  severe  reactions,  viz.,  chills,  fever,  vomiting,  diarrhea,  etc., 
but  such  symptoms  are  now  seen  only  exceptionally.  Symptoms  on 
the  part  of  the  nervous  system,  notably  in  connection  with  some  of 
the  cranial  nerves,  which  develop  in  relatively  rare  instances,  either 
during  the  first  few  days  or  only  after  several  months,  following 
the  use  of  salvarsan,  are  to  be  attributed  not  to  any  toxic  action 
on  the  part  of  the  drug,  but  to  the  localization  of  the  spirochetes, 
and  constitute  an  indication  for  the  further  use  of  the  drug  rather 
than  the  reverse  (see  Neurorelapses) . 

That  the  known  centra-indications  to  the  use  of  the  salvarsan 
should,  of  course,  be  considered  in  connection  with  every  case  goes 
without  saying  (see  below). 

Neosalvarsan. — Since  salvarsan  was  first  placed  upon  the  market, 
Ehrlich  has  attempted  to  modify  the  product  so  that  its  preparation 
for  injection  would  be  simplified  and  the  use  of  the  caustic  alkali, 
in  particular,  eliminated,  as  some  of  the  objectionable  features 
which  are  at  times  noted  after  the  injection  could  be  shown  to  be 
due  to  this  factor.  The  result  is  the  so-called  neosalvarsan. 

This  is  a  condensation  product  of  salvarsan  and  hydraldite  (for- 
maldehyde sulphoxylate  of  sodium),  the  reaction  taking  place 
according  to  the  equation: 


As  =  As  As  =  As 

/\    /\  /\    /\ 


NH2|  |NH2+HO.CH2.O.SONa 


OH     OH 


I 

OH    OH 


|NH.CH2.O.SONa+H20 


SALVARSAN  AND  ITS  USE  IN  TREATMENT  OF  SYPHILIS     269 

Like  salvarsan  the  new  product  is  a  yellow  powder,  which  is 
readily  soluble  in  water,  but  unlike  the  original  it  forms  a  neutral 
solution  so  that  no  addition  of  alkali  is  necessary  before  use.  As 
its  "initial"  toxicity  at  the  same  time  is  less  (1  gram  corresponding 
to  0.66  of  salvarsan),  and  its  spirillocidal  action  even  more  intense 
than  that  of  salvarsan,  still  better  results  may  be  anticipated  from 
its  use. 

The  solution  should  be  prepared  just  before  injection,  but  it  is  neces- 
sary to  use  a  salt  solution  of  lower  concentration,  i.  e.,  one  not 
stronger  than  0.4  per  cent.,  as  otherwise  it  will  be  turbid,  and  appar- 
ently more  toxic.  As  in  the  case  of  salvarsan,  moreover,  only  freshly 
distilled  or  sterile  water  should  be  used.  While  the  temperature  of 
the  saline  may  be  as  high  as  20°  C.,  the  liquid  should  not  be  heated 
after  solution  has  taken  place,  for  fear  of  causing  the  formation  of 
poisonous  oxidation  products.  For  the  same  reason,  it  is  recom- 
mended not  to  shake  the  solution  unnecessarily.  Older  solutions  turn 
a  reddish  color,  and  the  same  is  seen  in  the  case  of  the  powder 
itself,  if  the  ampoule  was  not  absolutely  air-tight;  such  preparations 
are  dangerous  and  should  be  discarded. 

For  injections,  0.6  to  1.5  grams  may  be  dissolved  in  200  to  250  c.c. 
of  saline  (for  dosage  see  below). 

Under  no  circumstances  should  the  neosalvarsan  be  used  sub- 
cutaneously  or  intramuscularly,  as  the  chances  of  its  oxidation  and 
hence  of  a  material  increase  in  its  toxicity  would  thus  be  much 
increased.  Nor  is  it  admissible,  even  if  the  drug  is  to  be  administered 
intravenously,  to  prepare  the  solution  in  bulk  for  several  patients, 
as  the  time  interval  elapsing  between  the  first  and  subsequent  injec- 
tions might  be  sufficient  to  cause  the  oxidation  of  the  drug.  The 
general  idea  underlying  the  administration  of  the  neosalvarsan  is 
thus  to  cut  down  the  time  exposure  of  the  drug  or  its  solution  to 
the  air  to  a  minimum. 

Reaction. — The  reaction  symptoms  which  follow  the  use  of  the 
neosalvarsan  are  usually  insignificant,  while  neglect  of  the  instruc- 
tions just  given  may  be  expected  to  produce  the  symptom-complex 
of  acute  arsenical  poisoning.  With  large  doses  exanthems  have  been 
observed  between  the  eighth  and  the  twelfth  day,  which  may  be 
avoided,  however,  if  smaller  amounts  are  given. 

Dosage  and  Frequency  of  Injection. — As  the  injection  of  any  spiril- 
locidal drug  that  does  not  effect  the  complete  destruction  of  all 


270  CHEMOTHERAPY 

the  parasites  at  a  single  dose  is  apt  to  lead  to  the  development  of 
serum  and  drug-fast  strains,  a  large  dose,  cceteris  paribus,  is  preferable 
to  a  small  dose;  if,  however,  a  large  dose  is  for  any  reason  not  advis- 
able, it  is  probably  best  to  inject  smaller  quantities  at  brief  intervals. 

While  0.5  gram  is  generally  recommended  as  the  initial  dose  of 
salvarsan  for  men,  and  a  slightly  smaller  amount  (0.3  to  0.4  gram) 
for  women,  some  investigators  have  used  larger  quantities  without 
observing  any  detrimental  effects.  In  subjects  that  are  not  in 
robust  health,  or  in  individuals  where  one  is  in  doubt  whether  to  use 
the  remedy  at  all,  it  is  best  to  give  a  small  initial  injection,  say  of 
0.2  gram,  and  to  repeat  this  dose  in  a  few  days  if  no  unusual  symp- 
toms develop.  In  young  babies  up  to  the  fourth  month  the  dose 
should  not  exceed  0.02  to  0.03  gram,  while  in  children  of  nine  or 
ten  years  of  age  0.1  and  0.2  gram  may  be  given,  which  may  be 
injected  into  the  gluteal  muscles,  the  amount  of  liquid  being,  of 
course,  proportionately  smaller.  The  pain  which  develops  after  the 
injection  may  be  controlled,  to  a  certain  extent  at  least,  by  hot 
compresses.  But,  as  I  have  pointed  out  above,  one  must  not  be 
surprised  if  local  necrosis  develops  after  the  use  of  the  remedy  in 
this  manner. 

If  the  neosalvarsan  is  to  be  employed,  1.5  gram  may  be  given  to 
men  and  1.2  gram  to  women,  but  it  is  recommended  not  to  start 
with  these  doses,  but  to  give  a  primary  injection  of  0.9  gram;  to 
allow  a  day  to  intervene  and  then  to  inject  1.2,  then  on  alternate 
days,  i.  e.,  with  a  day  of  rest  intervening,  1.35  gram  and  finally 
1 .5  gram.  That  the  dosage  can  be  pushed,  however,  is  shown  by  the 
fact  that  in  robust  men  6  grams  of  neosalvarsan,  corresponding  in 
arsenical  content  to  4  grams  of  salvarsan,  have  been  given  within 
seven  days. 

Babies  are  given  0.05  and  children  0.15  gram. 

Small  initial  doses  of  either  preparation  are  indicated  whenever 
there  is  reason  for  assuming  the  existence  of  syphilis  of  the  central 
nervous  system  and  its  meninges.  As  the  latter  are  known  to  be 
involved  quite  early  in  the  course  of  the  infection  already  Ehrlich 
suggests  that  in  early  secondary  cases  (especially  during  the  roseolar 
stage),  particularly  when  nervous  symptoms  of  any  kind  exist,  the 
average  initial  dose  (of  salvarsan)  should  not  be  higher  than  0.3  gram, 
and  that  in  suspicious  cases  it  may  be  best  to  begin  with  0.1  to  0.15 
gram.  This  dose  should  preferably  not  be  exceeded  even  at  the 


SALVARSAN  AND  ITS  USE  IN  TREATMENT  OF  SYPHILIS     271 

second  injection,  and  if  threatening  symptoms  exist  it  may  even 
be  better  to  begin  with  a  course  of  calomel  and  to  follow  this 
up  with  a  very  small  dose  of  salvarsan.  Our  final  aim,  however, 
should  be  the  injection  of  full  doses. 

Should  by  any  chance  symptoms  denoting  grave  danger  develop 
notwithstanding  this  mode  of  procedure,  lumbar  puncture  is  to  be 
performed  without  delay,  and  if  no  meningeal  involvement  then 
manifests  itself  (as  evidenced  by  the  presence  of  a  small  amount  of 
fluid  only),  this  is  to  be  followed  by  trephining.  Above  all  there 
should  be  no  delay  in  carrying  out  such  treatment. 

A  month  after  the  last  injection  the  Wassermann  test  is  made.  If 
this  still  shows  a  positive  reaction  the  salvarsan  treatment  should 
be  repeated,  and  may  advantageously  be  followed  by  a  brisk  course 
of  mercury.  If  the  latter  is  used,  a  month  should  then  elapse  during 
which  no  medication  whatever  is  employed,  before  the  next  test  is 
made,  and  so  on,  until  a  negative  reaction  is  reached.  From  this 
point  off  the  Wassermann  is  repeated  at  more  and  more  distant 
intervals  for  from  two  to  three  years  (see  Wassermann  Reaction). 

Contra-indications  to  the  Use  of  Salvarsan. — At  the  very  beginning 
of  its  use  Ehrlich  emphasized  the  importance  of  excluding  all  those 
cases  from  the  salvarsan  treatment  in  which  there  was  reason 
to  assume  the  existence  of  advanced  disease  of  the  heart  or  of 
the  central  nervous  system;  particularly  cases  of  angina  pectoris, 
aneurysm  (notably  of  the  cerebral  vessels),  advanced  paresis,  and 
cases  of  atrophy  of  the  optic  nerve,  while  in  other  syphilitic  diseases 
of  the  eyes  as  well  as  in  advanced  syphilis  of  the  abdominal  viscera 
the  remedy  may  be  advantageously  employed.  If  any  doubt  exists 
in  an  individual  case,  whether  the  remedy  should  be  used  or  not,  and 
the  condition  of  the  patient  so  far  as  the  syphilitic  process  in  itself 
is  concerned  makes  this  desirable,  a  careful  attempt  may  be  made 
with  a  very  small  dose  (0.1  gram),  which,  if  no  disturbing  symptoms 
develop,  may  then  be  followed  up  with  one  of  equal  size  or  even  a 
little  larger  (see  above,  Dosage). 

After  all  we  must  remember  that  the  number  of  deaths  which 
can  be  attributed  to  the  salvarsan  itself,  or  to  its  effect  upon  the 
syphilitic  process,  and  not  to  harmful  technique,  is  ridiculously 
small  in  comparison  with  the  enormous  number  of  cases  where  no 
harmful  result  has  followed  its  use,  and  where,  on  the  contrary,  the 
greatest  amount  of  good  has  been  accomplished.  As  Ehrlich  has 


272  CHEMOTHERAPY 

pointed  out,  the  toxicity  of  the  salvarsan  is  distinctly  less  than  that 
of  mercury. 

Neurorelapses. — Not  infrequently  certain  functional  disturbances 
have  been  noted  to  occur  in  connection  with  some  of  the  cranial 
nerves,  which  appear  very  soon  after  the  injection.  Ehrlich  is 
inclined  to  look  upon  these  as  corresponding  to  the  so-called  Herx- 
heimer  reaction,  which  is  so  frequently  observed  in  the  skin  soon 
after  the  use  of  salvarsan,  and  which  he  refers  to  the  liberation  of 
toxins  from  the  killed  spirochetes  and  to  their  local  irritating  effect. 
He  points  out  that  if  such  a  reaction  should  affect  one  of  the  cranial 
nerves  at  a  point  where  this  passes  through  a  narrow,  bony  canal, 
disturbance  in  function  would  be  a  very  probable  consequence, 
owing  to  swelling  and  resultant  compression.  Such  disturbances, 
however,  do  not  occur  within  a  few  hours  of  the  injection,  as  in  the 
case  of  the  true  Herxheimer  reaction,  affecting  the  skin,  but  only 
after  twenty-four  hours,  or  even  after  three  or  four  days,  as  the 
vascular  supply  of  the  nerves  is  but  little  developed,  and  a  longer 
time  must  elapse  before  a  sufficient  number  of  spirochetes  has  been 
killed  to  produce  a  local  reaction  of  moment.  Owing  to  the  same 
cause,  an  opportunity  is  afforded  in  these  localities  for  the  escape  of 
some  of  the  spirochetes  and  their  subsequent  development.  Should 
the  spirochetal  focus  be  very  small  in  comparison  to  the  size  of  the 
nerve  at  the  point  in  question,  so  that  no  pressure  would  result 
in  consequence  of  the  first  Herxheimer  reaction,  there  will,  of  course, 
be  no  occasion  for  the  development  of  acute  symptoms.  But  if, 
then,  the  surviving  spirochetes  increase  in  number  a  basis  would 
be  furnished  for  what  is  now  commonly  termed  a  neurorelapse. 

When  these  relapses,  which  usually  occur  two  or  three  months 
or  even  four  or  five  months  after  treatment,  were  first  observed, 
following  the  use  of  salvarsan,  they  were  attributed  to  the  contained 
arsenic  and  were  supposed  to  constitute  a  special  danger  attending 
its  use.  But  as  Ehrlich  has  pointed  out,  the  same  occurrences  have 
been  noted  in  connection  with  the  use  of  mercury,  and  to  judge  from 
the  collective  reports  of  Benario  they  are  no  more  frequent  after  the 
use  of  salvarsan  than  after  that  of  the  latter,  and  here  as  there  the 
same  nerves  are  especially  prone  to  attack,  viz.,  the  auditory,  the 
optic,  the  facial,  and  the  oculomotor,  while  the  fourth,  fifth,  sixth, 
and  twelfth  are  much  less  frequently  affected. 

Ehrlich  emphasizes  in  support  of  his  view  that  neurorelapses  only 


SALVARSAN  AND  ITS   USE  IN  TREATMENT  OF  SYPHILIS     273 

occur  during  that  period  of  the  disease  when  there  is  a  maximal 
distribution  of  spirochetes,  viz.,  during  the  early  secondary  stage, 
notably  in  connection  with  the  first  exanthem,  while  during  the  later 
stages  when  actual  nerve  lesions  exist  they  are  not  observed.  He 
regards  their  occurrence  as  evidence  of  a  nearly  complete  sterili- 
zation of  the  body,  and  very  aptly  compares  the  neurorelapse  to 
the  extensive  development  of  individual  bacterial  colonies  on  agar 
plates,  when  but  few  organisms  are  present,  as  contrasted  with  their 
tiny  size  when  the  number  is  large.  He  accordingly  advises  that 
such  cases  be  reinjected  with  the  salvarsan,  and  there  are  already  a 
number  of  reports  to  show  that  such  treatment  is  indeed  frequently 
followed  by  a  most  favorable  result,  while  it  is  well  known  that  a 
nerve  that  has  actually  been  damaged  by  arsenic  itself  (atoxyl 
for  example)  is  hopelessly  doomed  if  a  second  injection  is  given. 

Results. — While  there  is  evidence  to  show  that  the  therapia  magna 
sterilisans,  i.  e.,  the  complete  destruction  of  all  the  parasites  during 
a  single  course  of  treatment,  is  no  mere  dream  but  an  actual  possi- 
bility, this  point  is  not  reached  so  readily  in  syphilis  of  the  human 
being,  at  least,  as  in  the  spirillosis  of  chickens,  in  relapsing  fever, 
and  in  frambesia,  where  a  single  injection  is  usually  sufficient.  That 
a  cure  can  be  effected,  however,  in  a  relatively  short  time  is  beyond 
all  question. 

Here  as  elsewhere  in  the  treatment  of  disease  the  best  results  will 
be  obtained  if  this  is  instituted  early.  Gennerich  thus  reports,  that 
of  58  cases  of  primary  disease  that  had  been  treated  with  calomel, 
followed  by  salvarsan,  not  a  single  one  developed  secondaries,  nor 
did  the  Wassermann  reaction  become  positive  again,  and  that  of 
these,  20  had  already  been  followed  for  from  nine  to  sixteen  months 
at  the  time  of  writing.  Tanzer,  who  used  the  salvarsan  by  itself, 
similarly  reports  that  of  21  cases  which  could  be  followed  for  from 
three  to  thirteen  months,  none  had  a  relapse,  while  the  Wassermann 
reaction  remained  negative.  Arning  states  the  same  of  67  cases 
which  had  been  treated  with  salvarsan  and  mercury,  etc.  The  ques- 
tion, of  course,  might  rightfully  be  asked,  whether  these  people  could 
actually  be  regarded  as  cured,  and  whether  the  disease  had  not  merely 
become  latent.  Opposed  to  such  a  conclusion  is  the  fact  that  some 
cases  of  syphilis  which  had  been  treated  with  salvarsan,  and  in  whom 
no  further  symptoms  developed,  later  came  back  with  a  new  infection, 
i.  e.,  with  a  new  chancre,  which  would  prove  that  the  patient  must 
18 


274  CHEMOTHERAPY 

actually  have  been  cured,  since  reinfection  in  the  active  syphilitic 
is  not  possible.  To  this  it  might  be  objected  that  the  new  chancre 
was  in  reality  not  a  new  infection  but  a  relapse,  analogous  to  the 
neurorelapses  referred  to  above.  But  as  Ehrlich  remarks,  the  neuro- 
relapses  occur  after  a  period  of  from  two  to  five  months,  so  that 
if  no  symptoms  develop  within  that  period  of  time,  one  would 
hardly  be  justified  in  looking  upon  the  cases  referred  to  above  as 
being  latent. 

The  assumption  that  a  cure  had  actually  been  established  is,  how- 
ever, further  supported  by  the  fact  that  in  those  cases  which  could 
be  examined  in  this  direction  a  so-called  provocative  Wassermann 
reaction  could  not  be  elicited.  This  reaction  is  based  upon  the  idea 
that  in  individuals  in  whom  the  spirochetes  have  been  exterminated 
to  such  a  degree  that  a  positive  Wassermann  can  no  longer  be 
demonstrated  but  in  whom  a  small  number  of  organisms  still  remain, 
this  may  yet  be  done,  temporarily  at  least,  if  a  further  injection  of 
the  salvarsan  is  given,  or  if  a  few  large  doses  of  mercury  are  admin- 
istered. As  a  matter  of  fact,  it  can  be  shown  that  in  truly  latent 
cases  a  positive  Wassermann  may  indeed  be  obtained  in  this  manner 
in  the  course  of  two  weeks  from  the  time  of  the  test  treatment,  and 
Ehrlich  very  properly  advises  that  such  an  examination  should  be 
made  before  a  patient  is  finally  discharged  from  treatment. 

Further  evidence  of  the  remarkable  efficacy  of  the  salvarsan 
treatment  is  the  rapidity  with  which  the  spirochetes  disappear  from 
primary  sores  and  from  secondary  ulcerations.  This  usually  takes 
place  within  twenty-four  hours,  but  sometimes  even  more  rapidly. 
Schreiber  thus  mentions  a  case  that  had  been  treated  with  neo- 
salvarsan,  in  which  the  organisms  could  no  longer  be  demonstrated 
four  hours  after  the  injection.  He  nevertheless  advises  the  excision 
of  the  primary  sore  whenever  this  is  possible. 

While  the  best  results  may  thus  be  expected  during  the  primary 
stage  of  the  disease,  especially  if  several  doses  of  salvarsan,  possibly 
followed  by  mercury,  have  been  administered,  no  effect  whatever  is  to 
be  hoped  for  in  cases  that  are  no  longer  suffering  from  their  syphilis 
proper,  but  from  the  consequent  lesions.  Symptomatic  improve- 
ment, to  be  sure,  may  at  times  be  seen  even  then,  and  is  no  doubt 
due  to  the  destruction  of  the  few  foci  of  spirochetes  that  may  still 
be  remaining,  and  the  elimination  of  such  sources  of  toxin  production. 
But  upon  the  symptoms  that  are  the  outcome  of  the  actual  destruc- 


SALVARSAN  AND  ITS   USE  IN   TREATMENT  OF  SYPHILIS     275 

tion  of  important  cell  complexes,  such  as  the  blindness  and  ataxia 
of  tabes,  the  remedy  will  naturally  be  without  effect.  It  is  to  be 
noted,  however,  that  in  very  early  cases  one  may  occasionally  see 
remarkable  improvement  even  in  some  of  those  very  symptoms 
which  we  are  accustomed  to  refer  to  the  actual  destruction  of  nerve 
cells,  so  that  the  inference  suggests  itself  that  some  of  the  symptoms, 
of  tabes,  for  example,  may  be  due  both  to  toxic  influences  and  to  an 
actual  destruction  of  nerve  cells.  For  this  reason  the  remedy  may 
be  given  a  trial  in  tabes  and  paresis  at  the  first  sheet  lightning,  as 
Ehrlich  puts  it,  while  later  on  it  is,  of  course,  useless,  and  in  advanced 
paresis  especially,  its  employment  is  even  attended  by  a  certain 
amount  of  danger. 

As  the  destruction  of  spirochetes  leads  to  the  production  of  anti- 
bodies of  a  protective  character,  as  is  evidenced  by  the  beneficial 
effect  which  the  milk  of  salvarsan-treated  syphilitic  mothers  has  upon 
the  syphilitic  lesions  of  the  child,  it  has  been  suggested  to  extract 
blood  from  treated  syphilitic  patients  and  to  inject  the  serum  into 
the  subarachnoid  space  of  such  cases  of  cerebrospinal  syphilis 
(paresis)  in  which  salvarsan  is  of  no  benefit,  when  given  by  itself. 
But  as  yet  no  data  are  available  to  warrant  any  conclusions 
regarding  such  a  mode  of  procedure.  It  would  seem  logical,  but 
it  may  be  questioned  whether  the  injected  antibodies  would  reach 
the  spirochetes  in  sufficient  quantity  to  do  much  good. 

However  this  may  be,  if  we  eliminate  from  our  analysis  all  those 
cases  in  which  destructive  lesions  have  occurred,  and  sum  up  the 
findings  in  the  remainder,  there  is  overwhelming  evidence  to  show 
that  in  salvarsan,  either  by  itself  or  in  combination  with  mercury, 
we  have  a  treatment  by  which  we  cannot  only  produce  a  favorable 
influence  upon  the  clinical  symptoms,  but  actually  effect  a  cure, 
in  the  vast  majority  of  cases.  It  seems  very  doubtful  in  fact  whether 
any  cases  exist,  in  which  the  infection  cannot  be  completely  eradicated 
either  by  the  salvarsan  alone,  or  in  combination  or  alternation  with 
mercury,  if  the  results  of  the  treatment  are  controlled  at  frequent 
intervals  by  the  Wassermann  reaction,  and  if  the  treatment  itself 
is  carried  out  by  experts.  A  suitable  combination  of  the  syphilol- 
ogist's  clinical  knowledge  and  the  peculiar  training  of  the  immu- 
nologist  will  unquestionably  yield  the  best  results;  either  alone  is  not 
in  a  position  to  give  the  patient  the  very  best  that  can  be  given. 

To  enter  into  a  detailed  account  of  case  records  would  lead  us 


276  CHEMOTHERAPY 

too  far  afield,  however,  and  I  would  refer  those  who  are  interested  to 
the  special  literature  upon  the  subject;  suffice  it  to  say  at  this  place 
that  barring  those  cases  in  which  the  treatment  is  clearly  contra- 
indicated,  it  should  be  followed  whenever  there  is  reason  to  believe 
that  living  spirochetes  are  present  in  a  patient's  body,  as  evidenced 
either  by  the  character  of  the  clinical  symptoms  or  the  presence  of 
a  positive  Wassermann  reaction. 


SALVARSAN  AND  ITS  USE  IN  NON-SYPHILITIC  MALADIES 

While  salvarsan  has  gained  its  greatest  fame  in  the  treatment  of 
syphilis,  there  is  evidence  to  show  that  the  remedy  is  even  more 
effective  in  combating  other  infections  that  are  due  to  protozoan 
parasites.  It  has  thus  been  found  of  signal  value  in  the  treatment  of 
those  cases  of  tertian  malaria  which  are  refractory  to  quinine.  In 
this  connection  the  interesting  observation  has  been  made  that  in 
some  cases  of  this  order  the  administration  of  salvarsan  in  very 
small  doses  may  cause  the  refractory  behavior  to  quinine  to 
disappear. 

Brilliant  results  have  been  reported  by  many  observers  in  the 
treatment  of  relapsing  fever,  where  a  single  injection  suffices  to  cause 
the  parasites  to  disappear  and  to  effect  a  lasting  cure.  Equally 
favorable  results  have  been  obtained  in  frambesia,  which  plays  a 
more  important  role  among  the  plantation  workers  of  Surinam  than 
even  syphilis.  Koch  and  Flu  report  that  of  900  cases  which  had  thus 
been  treated  only  three  developed  a  relapse.  As  a  rule  a  single 
injection  is  sufficient,  or  at  most  two  injections.  Quite  important, 
further,  is  the  observation  of  Joannides  that  bilharziasis  can  be 
cured  with  a  single  injection.  The  same  is  reported  concerning 
the  effect  of  the  treatment  on  aleppo  boil,  while  it  seems  to  be  of 
no  avail  in  kala-azar.  Whether  or  not  the  remedy  is  of  use  in  the 
treatment  of  typhm  fever  is  not  yet  certain;  some  writers  report 
favorable  results,  while  others  are  less  enthusiastic.  In  amebiasis 
and  Vincent's  angina,  however,  it  seems  to  have  a  definitely  favor- 
able effect.  Wonderful  results  have  been  reported  in  yaws.  In 
the  treatment  of  sleeping  sickness  the  results  have  been  inconstant. 
As  the  tendency  to  the  development  of  arsenic-fast  strains  is  much 
greater  in  the  case  of  the  trypanosomes  than  in  the  spirochetes, 


CHEMOTHERAPY  IN  BACTERIAL  INFECTIONS  277 

every  attempt  should  here  be  made  to  destroy  the  parasites  with 
a  single  dose,  while  the  therapia  fractionata,  which  is  to  a  certain 
extent  permissible  in  syphilis,  is  less  apt  to  be  successful. 


CHEMOTHERAPY   IN   BACTERIAL   INFECTIONS 

While  the  application  of  the  new  science  of  chemotherapy  to  the 
study  of  protozoan  infections  has  thus  led  to  most  brilliant  results 
within  the  few  years  of  its  existence,  the  thought  naturally  suggests 
itself  whether  some  of  the  bacterial  infections  also  may  not  be  amen- 
able to  medicinal  treatment  upon  this  basis.  A  priori,  of  course, 
this  possibility  exists,  but  it  is  noteworthy  that  the  only  diseases  in 
which  a  specific  cure  could  be  effected  in  the  olden  days  of  medicine 
were  of  protozoan  origin,  i.  e.,  malaria  and  syphilis,  and  it  is  to  be 
feared  that  the  problems  are  much  more  complicated  in  the  bacterial 
infections.  There  is  some  evidence  to  show,  however,  that  here 
also  a  new  era  of  treatment  may  be  expected  to  dawn  in  the  near 
future  and  that  modern  pharmacology  when  approached  from  the 
standpoint  of  general  biology  may  succeed  in  accomplishing  what 
the  pharmacology  of  the  olden  days  failed  to  do.  A  more  intimate 
knowledge  of  cell  metabolism  and  above  all  of  cell  nutrition  will 
unquestionably  carry  with  it  the  solution  of  the  problem  of  bacterial 
infections.  At  this  place  I  would  only  briefly  refer  to  the  recent 
advances  in  the  chemotherapy  of  pneumococcus  infections.  Lamar 
has  thus  shown  in  the  animal  experiment  that  while  a  corresponding 
immune  serum  is  incapable  of  preventing  the  development  of  pneu- 
mococcus meningitis  when  introduced  subdurally  by  itself,  a  mixture 
of  sodium  oleate,  immune  serum  and  boric  acid  regularly  exerted 
a  more  powerful  influence  than  the  immune  serum  alone,  and  not 
only  prevented  the  occurrence  of  infection,  but  also,  when  adminis- 
tered separately,  arrested  the  progress  of  an  actually  established 
infection,  and  led,  often,  to  the  enduring  and  perfect  recovery  of 
the  animal. 

On  the  basis  that  a  certain  parallelism  in  their  biological  behavior 
exists  between  trypanosomes  and  pneumococci,  and  that  certain 
quinine  derivatives  were  found  to  have  a  trypanocidal  effect,  Morgen- 
roth  and  his  collaborators  undertook  corresponding  studies  in  animal 
infections  with  the  pneumococcus.  As  a  result  of  their  investiga- 


278  CHEMOTHERAPY 

tions  they  found  that  whereas  quinine,  hydroquinine,  and  hydro- 
chlorisoquinine  had  no  effect  upon  the  course  of  artificial  pneumo- 
coccus  infections,  ethyl-hydrocuprein  was  capable  of  arresting  the 
infection  in  50  per  cent,  of  their  animals,  when  given  six  hours  after 
the  inoculation,  i.  e.,  at  a  time  when  from  ten  to  a  thousand  multiples 
of  the  fatal  dose  of  organisms  were  already  in  circulation.  Although 
corresponding  experiments  in  the  human  being  have  not  as  yet 
yielded  encouraging  results,-  the  data  obtained  in  the  animal  experi- 
ment are  highly  significant,  as  they  have  clearly  demonstrated  that 
the  substance  in  question  has  a  selective  affinity  for  the  organisms 
concerned.  For  this  reason  it  would  seem  quite  within  the  domain  of 
possibility  that  a  substance  may  be  produced  which  may  be  more 
effective  and  freer  from  undesirable  side  effects,  or,  to  use  the  par- 
lance of  Ehrlich,  one  in  which  the  bacteriotropism  would  be  greatly 
predominating  over  its  organotropism. 


CHEMOTHERAPY  IN   MALIGNANT   DISEASE 

While  the  pathogenesis  of  malignant  disease  is  still  sub  judice, 
and  while  no  satisfactory  evidence  has  as  yet  been  furnished  to  sup- 
port the  belief  that  it  is  parasitic  in  origin,  it  would  seem  as  though 
the  principles  which  are  involved  in  its  non-surgical  treatment, 
are  after  all  the  same  as  those  with  which  we  have  become  familiar 
in  the  course  of  our  study  of  the  infectious  diseases  proper.  Here 
as  there  cells  are  multiplying  within  the  body,  which  in  malignant 
disease  are  in  a  manner  just  as  foreign  to  the  adjacent  normal  cells 
as  a  bacterium  or  a  protozoan  parasite  would  be,  and  here  as  there 
the  life  of  the  foreign  cell  in  its  abnormal  environment  leads  to 
disturbances  of  the  normal  functions  not  only  of  the  adjacent  tissues, 
but  of  the  body  at  large,  which  may  be  so  serious  in  character  that 
the  death  of  the  macroorganism  may  follow,  and  in  malignant 
disease  indeed  invariably  follows.  Here  as  there  then  the  main 
plan  of  our  treatment  must  be  to  so  influence  the  proliferating 
foreign  cell  as  to  lead  to  its  direct  destruction,  or  at  least  to  prevent 
its  development,  without  causing  undue  harm  to  the  normal  cells 
of  the  body.  Evidently  this  is  in  part  at  least  a  problem  of  modern 
pharmacology  and  one  which  is  intimately  connected  with  the  study 
of  immunity;  for  we  must  remember  that  cell  destruction  within 


CHEMOTHERAPY  IN  MALIGNANT  DISEASE  279 

the  body,  of  whatever  kind,  will  invariably  lead  to  a  response  on 
the  part  of  the  macroorganism  which  is  in  the  end  of  a  protective 
character.  It  may  accordingly  not  be  out  of  place  in  a  book  of  this 
character  to  briefly  review  some  of  the  more  promising  lines  of  inves- 
tigation in  the  domain  of  chemotherapy  which  are  at  the  present 
time  occupying  the  attention  of  students  of  the  cancer  problem. 
That  the  problem  should  be  intrinsically  more  difficult  in  malignant 
disease  goes  of  course  without  saying,  for  it  is  here  not  only  a  question 
of  finding  a  substance  which  would  have  a  low  grade  of  affinity 
for  the  body  cells  coupled  with  a  high  grade  of  affinity  for  a  cell 
which  is  a  perfect  stranger  to  the  macroorganism,  but  we  must 
actually  find  a  product  which  shall  have  a  high  affinity  for  a  type 
of  cell  which  after  all  is  a  product  of  the  same  organism  against 
whose  normal  cells,  so  to  speak,  its  affinity  must  be  low.  The  pos- 
sibility of  actually  finding  a  substance  of  this  character  will  naturally 
depend  upon  the  question  whether  the  proliferating  cell,  which  for 
want  of  a  better  term  we  may  speak  of  as  the  cancer  cell,  is  struc- 
turally and  functionally  identical  with  the  normal  cell  from  which 
it  has  originated,  or  whether  any  points  of  difference  exist  between 
the  two  types.  While  this  question  still  awaits  its  final  answer, 
we  may  say  that  there  is  some  evidence  to  show  that  points  of  differ- 
ence do  exist  between  the  normal  and  the  abnormal  cell,  and  that 
the  problem  is  hence  a  priori  not  a  hopeless  one. 

The  most  interesting  studies  which  have  a  bearing  upon  the 
possibility  of  effecting  a  cure  of  cancer  along  the  lines  of  modern 
chemotherapy  have  been  published  by  A.  v.  Wassermann  and  his 
collaborators. 

The  investigations  of  these  observers  were  based  upon  the  dis- 
covery by  Gosio  that  sodium  selenate  and  sodium  tellurate  are  more 
rapidly  reduced  by  cancer  cells  than  by  normal  cells,  and  that  this 
reduction  takes  place  within  the  bodies  of  the  cells.  Experiments  on 
tumor  mice  then  showed  that  the  injection  of  these  substances  into 
the  growths  may  actually  lead  to  their  complete  destruction.  The 
problem  now  was  to  devise  some  method  by  which  the  selenium  or 
the  tellurium  could  be  carried  to  the  tumor  through  the  circulation 
in  order  to  prove  that  the  substance  in  question  actually  possesses 
a  selective  affinity  for  the  tumor  cell,  and  remembering  also  that 
the  complete  eradication  of  the  malignant  growth,  excepting  in  the 
very  earliest  stage  of  the  disease,  can  scarcely  be  expected  by  means 


280  CHEMOTHERAPY 

of  local  or  even  regional  treatment.  Considering  the  meager  blood 
supply  of  epithelial  tumors  more  especially  the  idea  naturally 
suggested  itself  to  combine  the  selenium  or  tellurium  with  some 
substance  endowed  with  great  powers  of  diffusion,  and  to  utilize 
this  as  a  carrier  of  the  cell-destroying  metal.  As  eosin  possesses 
such  properties  this  was  selected  and  a  number  of  eosin-selenium 
compounds  tested  out  in  this  direction.  While  the  results  obtained 
were  not  uniform,  a  definite  cure  was  nevertheless  achieved  in  several 
animals,  and  with  the  demonstration  of  this  fact  the  cancer  prob- 
lem seems  to  have  been  solved  in  principle,  and  it  has  been  proved 
that  not  only  a  growing  tumor  can  be  caused  to  retrogress  entirely 
in  consequence  of  the  introduction  into  the  body  of  a  definite  chemical 
compound  by  the  intravenous  route,  but  also  that  this  is  possible 
without  causing  undue  harm  to  the  body  at  large. 

Of  the  modus  operandi  of  the  selenium  we  are  as  yet  in  ignorance. 
/   Neuberg  and  Caspari  have  expressed  the  opinion  that  the  injection  of 
certain  compounds  of  the  heavy  metals,  notably  when  administered 
in  colloidal  form,  favors  the  self-digestion  (autolysis)  of  the  tumor 
^\   cell,  and  they  succeeded  in  demonstrating  that  there  are  actually 
/  /   substances  which  possess  a  selective  affinity  for  cancer  cells,  and  that 
|    in  cancer  mice  it  is  possible  to  bring  about  the  dissolution  of  the  tumor 
with  such  "tumor  affinic"  substances.    They  tested  out  a  large  num- 
ber of  the  heavy  metals  and  could  verify  Wassermann's  observation 
that  selenium  also  possesses  such  properties.    They  found,  however, 
that  copper  and  tin  are  even  more  active  in  this  respect.     These 
experiments  have  given  rise  to  most  active  work  along  these  lines, 
and  sufficient  evidence  has  already  accumulated  to  warrant  the  hope 
that  ere  long  a  method  may  be  devised  which  may  be  applicable  in 
the  human  being  also.  Some  work  in  this  direction  is  indeed  already 
in  progress,  but  it  would  be  premature  to  draw  any  inferences  from 
the  little  that  has  as  yet  been  done. 

In  conclusion  we  would  yet  briefly  refer  to  the  work  of  R.  Werner 
and  his  collaborators.  Starting  from  a  certain  parallelism  which 
exists  between  the  effect  of  radium  and  x-ray  applications  and  the 
action  of  certain  cholin  salts  upon  the  leukocytes  and  the  reproductive 
glands,  and  the  fact,  moreover,  that  there  is  some  evidence  to  show 
that  the  lecithin  of  the  cells  is  decomposed  by  the  x-ray  and  radium 
emanations,  with  the  liberation  of  cholin,  these  investigators  ex- 
pressed the  opinion  that  the  beneficial  effects  of  radium  and  x-ray 


CHEMOTHERAPY  IN  MALIGNANT  DISEASE  281 

treatment  upon  malignant  growths  also  might  very  well  be  due  to 
the  liberation  and  action  of  these  very  substances.  They  accordingly 
studied  the  effect  of  various  cholin  preparations  upon  tumor  growth, 
and  found  as  a  matter  of  fact  that  the  changes  resulting  in  the  tumors 
were  perfectly  analogous  to  those  produced  by  ray  treatment. 
Since  cholin  could  be  shown  to  possess  a  high  power  of  diffusion 
the  thought  then  suggested  itself,  in  view  of  the  observations  above 
related  concerning  the  selective  action  of  colloidal  metals  upon  tumor 
cells,  to  use  the  cholin  as  carrier  of  these  metals.  Studies  in  this 
direction  showed  that  beneficial  effects  may  indeed  be  thus  achieved 
in  the  animal  experiment  with  colloidal  selenium,  copper,  cobalt, 
iron,  zinc,  and  arsenic,  when  dissolved  in  cholin  solutions,  and  that 
this  could  be  accentuated  by  combining  a  metal  of  high  electrical 
tension  with  one  of  low  tension,  or  even  with  one  which  in  itself 
is  inactive,  such  as  selenium-vanadium,  selenium-cobalt,  copper- 
cobalt,  etc.  There  is  thus  still  another  avenue  open  for  attack, 
and  one  which  likewise  seems  to  promise  profitable  results. 


CHAPTER  XV 

THE  APPLICATION  OF  IMMUNOLOGICAL 
PRINCIPLES  TO  DIAGNOSIS 

WHILE  in  the  foregoing  chapters  we  have  been  interested  largely 
in  the  reaction  of  the  animal  body  to  the  introduction  of  alien  cells 
and  cell  products,  from  the  standpoint  of  therapy,  it  is  important 
to  note  that  some  of  the  principles  involved  in  these  reactions  have 
also  found  application  in  the  diagnosis  of  many  of  the  infectious 
diseases.  The  recognition  of  the  formation  of  agglutinins  has  thus 
led  to  the  discovery  of  the  most  important  method  in  the  diagnosis 
of  typhoid  fever,  paratyphoid  fever,  and  Malta  fever;  the  principle 
underlying  the  formation  of  bacteriolysins  is  utilized  in  the  diagnosis 
of  cholera;  the  formation  of  special  antibodies,  which  in  the  presence 
of  corresponding  antigen  absorb  complement,  and  which  may  thus 
be  recognized  indirectly  by  the  demonstration  of  such  complement 
fixation,  serve  as  a  basis  of  the  Wassermann  diagnosis  of  syphilis;  the 
recognition  of  the  general  allergic  state  in  the  sense  of  v.  Pirquet  has 
led  to  some  of  the  most  important  methods  in  the  diagnosis  of  tuber- 
culosis and  syphilis;  the  precipitins  play  an  important  role  in  the 
recognition  of  specific  albumins,  and  serve  as  a  basis  of  the  modern 
tests  for  blood  in  legal  medicine;  the  formation  of  antiferments  has 
been  utilized  in  the  diagnosis  of  cancer,  etc. 

While  a  detailed  account  of  all  the  immunological  methods  of 
diagnosis  would  lead  us  too  far,  and  would  indeed  furnish  sufficient 
material  for  a  special  volume,  it  may  not  be  out  of  place  to  consider 
a  few  of  the  more  important  methods  of  this  order  in  some  detail. 


THE    AGGLUTINATION   REACTION 

In  1896  Gruber  and  Durham  pointed  out  that  the  addition  of 
cholera  and  colon  immune  serum  to  bouillon  cultures  of  the  corre- 
sponding organisms  produced  a  remarkable  effect,  for  on  standing  for 


THE  AGGLUTINATION  REACTION  283 

a  number  of  hours  the  turbidity  of  the  cultures  disappeared;  while 
all  the  bacteria  had  settled  to  the  bottom.  This  peculiar  behavior, 
as  we  now  know,  is  owing  to  the  presence,  in  the  sera  in  question,  of 
certain  antibodies  known  as  agglutinins  which  are 'formed  as  a  result 
of  infection  (sc.,  immunization),  and  are  characterized  by  the  fact 
that  in  suitable  dilution  they  will  cause  the  arrest  of  motility  and 
agglutination  of  the  corresponding  organisms  (see  also  section  on 
Antibodies) .  Normal  serum,  it  is  true,  will  also  do  this  to  a  certain 
extent,  but  only  when  used  in  a  fair  degree  of  concentration,  and 
then  only  imperfectly,  while  with  immune  sera  the  complete  reaction 
may  be  obtained  even  though  the  serum  be  diluted  many  times. 
In  this  sense  the  reaction  is  specific,  and  may  be  employed  both  for 
the  identification  of  a  given  organism  as  also  for  the  recognition 
of  the  nature  of  an  immune  serum.  In  the  first  instance  an  emul- 
sion of  the  unknown  bacterium  is  brought  together  with  diluted 
test  sera,  corresponding  to  those  organisms  which  would  enter  into 
consideration  from  a  diagnostic  standpoint.  If,  then,  the  bacterium 
in  question  is  agglutinated  by  an  antityphoid  serum,  for  example, 
but  not  by  an  anticolon  or  an  antidysentery  serum,  the  inference 
would  be  (within  certain  experimental  limitations)  that  we  are 
dealing  with  the  typhoid  bacillus.  On  the  other  hand  the  unknown 
serum,  in  a  certain  degree  of  dilution,  is  tested  against  a  series  of 
organisms,  when  a  positive  result  with  one  of  these  would  indicate 
the  nature  of  the  infection.  From  both  standpoints  the  agglutina- 
tion reaction  has  thus  a  wide  sphere  of  application. 

Very  soon  after  the  discovery  of  Gruber  and  Durham,  Widal 
found  that  the  formation  of  agglutinins  in  typhoid  fever  begins 
quite  early  in  the  course  of  the  disease,  i.  e.,  at  a  time  when  from 
the  usual  symptoms  the  diagnosis  cannot  as  yet  be  made  with  cer- 
tainty, and  he  thus  established  a  method  of  diagnosis  which  in  some 
one  of  its  numerous  technical  modifications  is  now  used  the  world 
over,  and  is  generally  known  as  the  Widal  reaction.  Further  studies, 
then,  showed  that  the  formation  of  agglutinins  in  other  infections 
likewise  begins  while  the  disease  is  in  actual  progress,  and  that  the 
same  principle  may  be  successfully  utilized  for  diagnostic  purposes  in- 
a  number  of  other  maladies  besides  typhoid  fever.  This  is  notably 
the  case  in  paratyphoid  infections,  in  Malta  fever,  and  in  meningo- 
coccus  infections.  In  other  maladies  agglutinin  formation  also  takes 
place,  but  either  does  not  begin  early  enough  to  be  of  service  in 


284  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

diagnosis  (as  in  plague,  for  example),  or  there  are  certain  technical 
difficulties  which  make  it  inapplicable  (tuberculosis,  cholera),  while 
in  still  others  a  diagnosis  can  be  conveniently  reached  in  an  even 
more  direct  manner  (as  by  the  isolation  and  cultivation  of  the 
offending  microorganism),  etc. 

To  give  a  general  idea  of  the  technical  method  of  procedure  it  will 
be  best  to  describe  the  reaction  as  it  is  applied  to  the  diagnosis 
of  typhoid  fever,  i.  e.,  the  Widal  reaction  proper. 

The  Widal  Reaction. — TECHNIQUE. — Microscopic  Method. — A  small 
amount  of  blood  (5  to  10  drops)  is  collected  in  a  little  glass  tube  or  in 
one  of  the  capsules  pictured  in  Plate  III.  The  serum  is  separated  by 
centrifugation  and  a  drop  diluted  in  the  white  mixing  pipette  accom- 
panying the  hemocytometric  counting  chamber  in  the  proportion  of 
1  to  20.  From  this,  subdilutions  of  1  to  40,  1  to  80,  1  to  160  are  pre- 
pared with  the  aid  of  the  same  pipette,  normal  salt  solution  being 
used  as  diluent  in  all  cases.  Four  slides  are  then  ringed  with  vaseline, 
and  into  each  little  chamber  a  drop  of  the  diluted  serum  is  placed 
together  with  a  drop  of  a  typhoid  culture  in  bouillon,  not  more  than 
twenty-four  hours  old.  The  resultant  dilutions  will  then  be  1  to  40, 
1  to  80,  1  to  160,  1  to  320.  A  cover-glass  is  adjusted  so  as  to  be  in 
contact  all  around  with  the  vaseline,  as  also  with  the  drop  in  the  cen- 
tral chamber.  The  specimens  are  immediately  examined  with  the 
middle  power  of  the  usual  microscopic  outfit  (J  B.  and  L.;  No.  6  or  7 
Leitz),  and  discarded  if  any  large  clumps  of  bacteria  are  seen. 
Should  this  be  the  case,  it  is  well  to  make  new  mounts  with  a  culture 
that  has  been  centrifugalized  for  a  minute  or  two,  the  supernatant 
fluid  only  being  used,  in  which  no  clumps  will  be  found.  If  the 
mount  is  satisfactory,  it  is  set  aside  and  reexamined  at  the  expira- 
tion of  half  an  hour.  If  the  reaction  is  positive,  all  the  bacilli  will 
be  found  motionless  at  the  expiration  of  this  time,  and  gathered  in 
clumps  of  variable  size  (Fig.  15).  This  will  be  the  case  at  least  in 
the  lowest  dilutions,  while  in  the  higher  ones  it  may  be  necessary 
to  wait  until  another  half-hour  has  expired.  The  higher  the  dilution 
in  which  complete  clumping  may  be  obtained  the  greater  is  the 
diagnostic  significance  of  the  reaction.  Ordinarily,  complete  clump- 
ing at  the  end  of  half  an  hour  in  a  dilution  of  1  to  40  is  sufficient;  if, 
however,  the  question  of  paratyphoid  enters  into  consideration,  the 
result  in  the  higher  dilutions  should  be  considered.  As  the  typhoid 
and  paratyphoid  bacilli  carry  certain  receptors  in  common,  the 


THE  AGGLUTINATION  REACTION 


285 


corresponding  antisera  also  will  contain  certain  agglutinins  in 
common  which  can  unite  with  both  types  of  organisms,  and  thus 
give  rise  to  agglutination  in  the  lower  dilutions.  As  the  more 
specific  receptors,  however,  predominate  over  those  that  are  com- 
mon to  both  types,  the  corresponding  agglutinins  will  also  be 
more  abundant  in  the  antisera,  so  that  the  type  of  infection  can  be 
established  from  the  higher  dilutions  in  which  a  serum  will  cause 
agglutination  of  a  given  organism. 


FIG.  17 


Positive  agglutination  reaction. 


A  material  advance  in  the  practical  applicability  of  the  Widal 
reaction  was  achieved  when  it  was  discovered  that  it  is  not  necessary 
to  work  with  living  cultures  of  the  typhoid  bacillus,  but  that  dead 
organisms  will  answer  just  as  well,  providing  that  the  strain  was 
readily  agglutinable  before  being  killed.  To  this  end  it  is  convenient 
to  prepare  a  bouillon  culture  in  an  Erlenmeyer  flask,  to  incubate 
for  twenty-four  hours  at  37°  to  40°  C.,  and  then  to  add  40  per  cent, 
formalin  solution  (i.  e.,  the  concentrated  solution  of  the  Pharma- 
copoeia) to  the  extent  of  1  per  cent.  After  standing  for  two  to  five 
days  in  the  incubator  the  emulsion  is  centrifugalized,  the  bacilli 
are  washed  with  two  changes  of  sterile  normal  salt  solution  and 
diluted  to  the  original  volume,  when  the  fluid  emulsion  may  be 


286  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

preserved  in  sterile  glass  beads  in  the  ice-box.  In  this  manner  the 
material  will  keep  for  months,  and  can  be  used  either  for  the  micro- 
scopic test,  in  which  case  the  time  of  examination  should  be  extended 
'  to  two  hours,  or  it  may  be  employed  in  the  macroscopic  test  described 
below. 

Macroscopic  Method. — This  method  is  just  as  exact  as  the  micro- 
scopic method,  and  is  in  a  manner  less  apt  to  lead  to  confusion; 
somewhat  larger  amounts  of  blood,  however,  are  required  (1  c.c.). 
The  serum  is  diluted  in  the  same  proportions  as  described  above. 
Equal  quantities  (0.25  c.c.)  are  then  placed  in  small  tubes,  such  as 
the  collecting  tubes  figured  in  Plate  III,  and  treated  with  equal 
volumes  of  the  bacterial  emulsion.  These  tubes  together  with  a 
control  of  equal  volumes  of  saline  and  bacterial  emulsion  are  placed 
in  the  incubator,  or  some  other  warm  place,  and  are  examined  after 
twelve  to  twenty-four  hours.  If  the  reaction  is  positive  the  bacteria 
in  the  serum  tubes  will  have  settled  to  the  bottom,  leaving  the  super- 
natant fluid  almost  clear,  thus  contrasting  sharply  with  the  control 
which  is  still  as  turbid  as  it  was  in  the  beginning. 

RESULTS. — While  a  positive  Widal  reaction  may  be  obtained  as 
early  as  the  first  day  of  the  disease,  meaning  thereby  the  first  day 
that  the  patient  spends  in  bed,  or  the  fifth  of  general  malaise,  such 
an  occurrence  must  be  viewed  as  a  great  rarity.  In  the  vast  majority 
of  cases  a  positive  result  is  obtained  only  after  the  fifth  or  sixth  day  in 
bed.  As  the  likelihood  of  positive  bacteriological  findings  is  greatest 
during  the  first  week  of  the  disease,  an  examination  in  this  direction 
may  at  this  time  well  take  precedence  over  the  agglutination  test. 
During  the  second  week,  when  the  value  of  the  two  methods  is  on 
a  par,  convenience  may  decide  which  one  is  to  be  employed.  After 
this,  however,  the  agglutination  test  should  be  given  the  preference. 
Experience  has  shown  that  a  positive  reaction  may  be  obtained  in  prac- 
tically all  cases  of  true  typhoid  fever,  but  it  is  clear  from  what  has 
been  said  that  much  depends  upon  the  period  of  the  disease  at  which 
the  examination  is  made.  The  production  of  agglutinins  evidently 
does  not  begin  at  the  same  time  in  all  cases,  and  does  not  become 
fully  established  until  after  the  disease  has  progressed  for  a  certain 
length  of  time.  It  may  happen,  indeed,  that  a  positive  reaction 
is  not  obtained  until  convalescence,  or  even  until  a  subsequent 
relapse  occurs.  For  this  reason  it  is  advisable  to  repeat  the  examina- 
tion at  frequent  intervals,  if  on  first  trial  a  negative  result  is  obtained. 


THE  AGGLUTINATION  REACTION  287 

Intermittence  of  the  reaction,  moreover,  is  quite  common  and  empha- 
sizes the  necessity  of  frequent  examinations  still  farther. 

While  in  some  instances  the  reaction  disappears  very  soon  after  the 
temperature  has  returned  to  normal,  and  even  earlier,  it  generally 
continues  well  into  convalescence,  and  may,  in  some  instances,  be 
obtained  after  months  and  even  years  following  the  attack.  In  a 
series  of  71  post-typhoid  cases,  Krause  found  the  reaction  in  36, 
viz.,  in  16  of  26  cases  examined  within  a  year,  in  12  of  21  examined 
between  the  second  and  the  fifth  year,  in  7  of  19  between  the  fifth 
and  the  tenth,  and  in  1  case  out  of  5  between  the  tenth  and  twentieth 
(twelfth)  year.  In  three  instances  no  reaction  could  be  obtained 
within  a  month  of  the  disease.  To  what  extent  the  continued 
presence  of  typhoid  agglutinins  may  be  referable  to  the  persistence 
of  the  corresponding  bacilli  in  the  body  has  not  been  ascertained.  It 
is  known  that  they  may  persist  in  the  gall-bladder  and  in  the  urinary 
bladder  for  a  long  time,  and  in  several  instances  they  have  been  found 
where  no  history  of  an  antecedent  typhoid  fever  could  be  obtained. 
In  a  case  of  cholelithiasis,  reported  by  Gushing,  typhoid  bacilli  were 
found  in  the  gall-bladder,  and  distinct  clumping  obtained  with  a 
dilution  of  1  to  30,  although  the  individual  gave  no  history  of  typhoid 
whatsoever.  Cases,  further,  are  occasionally  seen  which  clinically 
resemble  typhoid  fever  very  closely,  but  which  do  not  give  the  Widal 
reaction  at  any  time,  with  the  usual  dilution  of  1  to  50.  Some  of 
these  cases  are  referable  to  infection  with  organisms  which  are  closely 
related  to  the  typhoid  bacillus  and  which  also  give  rise  to  the  forma- 
tion of  agglutinins.  These,  however,  do  not  react  with  the  typhoid 
bacillus  excepting  in  low  dilution. 

Infection  with  related  organisms  may  also  be  responsible  for  certain 
cases  of  febrile  jaundice  (Weil's  disease),  in  which  agglutination 
of  the  typhoid  bacillus  has  been  observed.  In  others  the  reaction 
may  be  due  to  a  localized  infection  with  typhoid  bacilli.  The  biliary 
constituents  in  any  event  are  not  responsible  for  the  reaction.  This 
is  clear  from  the  observation  of  Kammerer,  who  obtained  agglutina- 
tion in  only  3  cases  of  jaundice  out  of  50  selected  at  random. 

In  the  diagnosis  of  paratyphoid,  Malta  fever,  and  meningococcus 
infections  a  corresponding  technique  is  employed,  for  a  consideration 
of  which  the  reader  is  referred  to  special  works  dealing  with  diag- 
nostic methods  from  the  laboratory  standpoint. 


288  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 


BACTERIOLYTIC   REACTIONS 

It  will  be  recalled  that  Pfeiffer  pointed  out  that  cholera  vibrios 
when  introduced  into  the  peritoneal  cavity  of  cholera-immune 
guinea-pigs  are  there  rapidly  destroyed  through  the  agency  of  the 
normal  complement  of  the  animal  and  the  bacteriolytic  amboceptor 
which  has  been  produced  in  consequence  of  immunization.  The 
principle  underlying  this  reaction  has  been  recommended  for  the 
diagnosis  of  both  cholera  and  typhoid  fever,  but  is  at  present  only 
utilized  in  connection  with  the  former  malady,  whereas,  owing  to 
its  greater  simplicity,  the  agglutination  test  is  almost  exclusively 
employed  in  typhoid  fever.  This  latter  test,  as  we  have  already  seen, 
is  for  technical  reasons  inapplicable  in  the  diagnosis  of  cholera. 

As  in  the  case  of  the  agglutination  test,  Pfeiffer's  reaction  also 
can  be  utilized  either  for  the  purpose  of  identifying  the  organism 
in  question,  or  in  searching  for  the  corresponding  amboceptor  in 
the  serum  of  a  patient. 

In  the  first  instance  the  organism  under  consideration  is  suspended 
in  cholera  immune  serum,  and  the  mixture  injected  into  the  peritoneal 
cavity  of  a  guinea-pig,  when  the  prompt  occurrence  of  bacteriolysis 
will  indicate  that  the  organism  was  actually  the  cholera  vibrio.  In 
the  second  case  the  serum  to  be  tested  is  inoculated  with  cholera 
vibrios  and  likewise  injected  into  a  guinea-pig,  when  the  occurrence 
of  bacteriolysis  will  prove  that  the  serum  contained  anticholera 
amboceptors  and  was  hence  derived  from  an  individual  who  must 
recently  have  passed  through  an  attack  of  the  illness  in  question. 

It  goes  without  saying,  of  course,  that  serum  and  organisms  must 
in  both  instances  be  combined  in  certain  definite  proportions,  to 
which  end  the  following  procedure  may  be  employed,  as  recom- 
mended by  the  Prussian  Institute  for  Infectious  Diseases. 

1.  Pfeiffer's  Test  as  Applied  to  the  Identification  of  Cholera  Vibrios. 
— For  this  purpose  an  anticholera  rabbit  serum  should  be  available 
which  should  be  of  such  strength  at  least  that  0.0002  gram  will  cause 
the  complete  destruction  of  an  oese  ( =  2  milligrams)  of  an  eighteen- 
hour-old  agar  culture  of  the  cholera  vibrio  within  one  hour  after 
injection  into  the  peritoneal  cavity  of  a  guinea-pig. 

One  pig  (^4)  is  then  injected  with  five  times  the  titer  dose  of  the 
immune  serum^L  e.,  1  milligram  together  with  one  oese  (  =  2  miffi- 


'A)  is  1 
'inn,  L 

<r 


BACTERIOLYTIC  REACTIONS  289 

grams)  of  an  eighteen-hour-old  culture  of  the  suspected  organism, 
suspended  in  1  c.c.  of  broth.  A  second  animal  (B)  is  given  ten  titer 
doses  (  =  2  milligrams)  with  the  same  quantity  of  organisms.  A 
third  (C)  receives  50  multiples  of  the  titer  dose,  i.  e.,  10  milligrams, 
of  normal  serum,  however,  but  taken  from  an  animal  of  the  same 
species  as  that  furnishing  the  immune  serum,  together  with  the  same 
quantity  of  organisms  as  A  and  B,  while  a  fourth  guinea-pig  ( D)  is 
injected  with  the  same  dose  of  bacteria,  but  without  any  serum. 
The  animals  should  all  be  of  about  the  same  weight  (250  grams), 
and  are  all  injected  intraperitoneally.  To  this  end  it  is  recommended 
to  make  a  small  incision  through  the  skin  and  to  inject  through  a 
cannula  with  a  blunt  point.  By  the  aid  of  glass  capillaries  a  droplet 
of  the  peritoneal  fluid  is  then  procured  through  the  same  incision, 
immediately  after  the  injection,  a  second  one  twenty  minutes  later, 
and  a  third  one  at  the  expiration  of  one  hour.  The  specimens  are 
examined  as  hanging  drops  with  an  oil-immersion  lens.  If  the 
organism  under  consideration  is  the  cholera  vibrio,  typical  granule 
formation  and  lysis  will  be  observed  in  specimens  A  and  B  after 
twenty  minutes  already,  and  at  the  latest  at  the  expiration  of  one 
hour;  while  in  C  and  D  there  will  be  large  numbers  of  actively  motile 
organisms  or  such  at  least  in  which  the  form  has  been  well  preserved, 
the  C  animal  being  the  control  to  A  and  B.  The  object  of  injecting 
D  is  merely  to  prove  that  the  organism  in  question  is  virulent  and 
this  animal  as  well  as  C,>  of  course,  should  die,  while  A  and  B  remain 
alive.  If  the  result  then  turns  out  as  just  indicated,  the  inference 
is  justifiable  that  the  organism  under  examination  was  really  the 
cholera  vibrio. 

2.  Pfeiffer's  Test  as  Applied  to  the  Recognition  of  Recent  Cholera 
Infections. — In  this  case  the  individual's  serum  is  diluted  with  broth 
in  the  proportion  of  1  to  20,  1  to  100,  and  1  to  500,  when  guinea-pigs 
are  each  inoculated  as  described  above  with  1  c.c.  of  various  dilutions, 
together  with  one  oese  ( =  2  milligrams)  of  an  eighteen-hour-old  agar 
culture  of  a  virulent  cholera  strain.  If  extensive  bacteriolysis  can 
then  be  demonstrated  at  the  expiration  of  twenty  minutes,  or  at  most 
an  hour,  the  inference  is  justifiable  that  the  person  has  recently 
passed  through  an  attack  of  cholera. 

PREPARATION  OF  THE  CHOLERA  IMMUNE  SERUM. — The  cholera 
immune  serum  which  is  required  in  test  1  (above)  is  prepared  as 
follows:  A  number  of  rabbits  are  each  injected  intraperitoneally 
19 


290 


IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 


with  a  single  cholera  agar  culture  that  has  been  killed  by  exposure 
for  one  hour  to  a  temperature  of  56°  C.  Two  weeks  later  the  animals 
are  bled  to  death,  the  sera  mixed,  evaporated  in  a  vacuum,  and  the 
dry  residue  is  put  up  in  portions  of  0.1  or  0.2  gram,  in  glass  beads, 
which  are  then  sealed.  The  titer  of  this  preparation  must  be  ascer- 
tained before  use;  it  usually  corresponds  to  about  0.0005  milligram 
as  calculated  for  the  liquid  serum. 

In  lieu  of  the  bacteriolytic  test  in  vivo,  as  outlined  above,  Stern 
and  Korte  have  proposed  a  corresponding  method  of  examination 
in  vitro,  for  a  consideration  of  which  the  reader  is  referred  to  special 
works. 


DIAGNOSTIC  REACTIONS  DEPENDING  UPON  COMPLEMENT 

FIXATION 

When  reaction  products  of  amboceptor  character  are  brought 
together  with  their  corresponding  antigens  in  the  presence  of  com- 
plement, an  interaction  takes  place  between  the  two  first  mentioned 
bodies,  in  consequence  of  which  complement  is  bound.  This  can  be 
demonstrated  by  the  subsequent  addition  of  washed  red  corpuscles, 


Antigen 


FIG.  18 


10 


Syphilitic 
Amboceptor 


10 


Complement 


Hemolytic 
Amboceptor 


Schema  illustrating  the  principle  of  the  Wassermann  reaction. 


and  a  corresponding  hemolytic  amboceptor,  when  hemolysis  will 
either  not  occur  at  all,  or  does  so  only  to  a  limited  extent,  according 
to  the  degree  to  which  the  available  quantity  of  complement  has  been 
bound.  The  exact  outcome  of  the  reaction  will,  of  course,  depend 
upon  quantitative  conditions.  If  we  suppose  that  the  interaction 


DIAGNOSTIC  REACTIONS  291 

between  the  various  components  takes  place  according  to  units, 
it  is  clear  that  if  ten  units  of  antigen,  for  example,  were  to  combine 
with  ten  units  of  the  corresponding  antibody,  and  if  ten  units  of 
complement  were  absorbed,  then  upon  the  subsequent  addition  of 
ten  units  of  corpuscles  and  ten  units  of  hemolytic  amboceptor  no 
hemolysis  whatever  could  take  place. 

If  no  antibody  corresponding  to  the  antigen  were  present,  the 
ten  units  of  complement  would  remain  free,  and  could  then  combine 
with  the  ten  units  of  the  hemolytic  amboceptor,  in  which  case 
complete  hemolysis  of  the  ten  units  of  red  cells  would  take  place. 
Between  these  two  extremes,  various  grades  of  hemolysis  are,  of 
course,  possible,  according  to  the  quantity  of  antibody  that  is  present. 

This  reaction,  like  the  agglutination  reaction  and  the  Pfeiffer  reac- 
tion, can  be  used  both  for  the  purpose  of  identifying  a  given  organism 
as  also  for  demonstrating  the  presence  or  absence  of  certain  ambocep- 
tors  in  the  blood  serum.  The  recognition  of  this  fact  led  to  the  dis- 
covery that  in  syphilis,  antibodies  appear  in  the  serum  which  are 
different  from  the  common  bacteriolytic  amboceptors,  insofar  as  they 
will  combine  with  substances  that  are  normal  constituents  of  the 
body,  i.  e.,  certain  lipoids.  Between  the  latter  and  the  corresponding 
syphilitic  antibody,  however,  an  analogous  reaction  takes  place,  as 
between  bacteria  and  their  amboceptors,  in  consequence  of  which 
complement  is  absorbed,  so  that  the  same  principle  can  be  utilized 
in  the  diagnosis  of  syphilitic  infections  as  well.  Applied  to  this  end, 
the  reaction  is  spoken  of  as  the  Wassermann  reaction,  as  Wassermann 
was  the  first  to  purposely  employ  the  principle  as  originally  under- 
stood, to  the  diagnosis  of  the  infection  in  question.  The  discovery 
of  this  reaction  must  rank  as  one  of  the  most  important  in  the  history 
of  medicine,  and  in  its  absence  the  triumphs  of  Ehrlich's  salvarsan 
could  never  have  been  achieved.  Its  employment,  as  a  matter  of 
fact,  forms  the  basis  of  the  modern  treatment  of  syphilis,  and  serves 
as  the  most  delicate  indicator  of  the  resultant  changes  which  lead  to 
the  recovery  of  the  patient,  besides  being  the  most  delicate  method 
that  we  possess  for  the  diagnosis  of  latent  syphilitic  lesions. 

The  Wassermann  Reaction. — When  Wassermann  first  applied  the 
principle  of  complement  fixation  to  the  study  of  syphilitic  patients 
his  idea  was  that  antibodies  of  amboceptor  character  might  be 
present  in  the  blood  serum  of  such  individuals,  in  which  case  it  should 
be  possible  to  demonstrate  these  by  bringing  them  together  with 


292  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

spirochetal  antigen  on  the  one  hand  and  complement  on  the  other. 
As  shown  in  Fig.  18,  the  complement  would  then  be  bound  to  a 
greater  or  less  extent  as  the  result  of  the  interaction  between  the 
other  two  factors.  Since  the  cultivation  of  the  syphilitic  spirochete 
had  at  that  time  not  been  accomplished,  however,  Wassermann  was 
obliged  to  make  use  of  extracts  of  organs  which  were  rich  in  the 
organisms  in  question.  To  this  end  he  employed  saline  extracts 
of  livers  from  syphilitic  fetuses.  With  such  material  as  antigen 
he  then  actually  obtained  complement  fixation  of  marked  degree, 
and  he  very  naturally  concluded  that  the  reaction  which  took  place 
was  one  corresponding  to  that  occurring  between  a  bacteriolytic 
amboceptor  and  its  corresponding  antigen. 

Later  studies,  however,  showed  that  this  could  not  be  the  case, 
since  identical  results  were  obtained  with  alcoholic  extracts  derived 
not  only  from  syphilitic  organs,  but  from  perfectly  normal  tissues 
as  well.  At  the  present  time  we  know  that  the  reacting  substance 
of  the  "antigen"  is  in  no  sense  a  specific  constituent  of  the  spirochete, 
but  apparently  a  lipoid  of  the  order  of  lecithin.  The  syphilitic  anti- 
body accordingly  cannot  be  an  amboceptor  in  the  sense  of  Ehrlich,  but 
is  evidently  a  substance  which  possesses  a  marked  affinity  for  certain 
lipoidal  bodies,  with  which  it  is  capable  of  interacting,  with  the 
consequent  absorption  of  complement.  Of  the  nature  of  this  inter- 
action we  know  nothing.  Pending  investigations  in  this  direction 
we  may  nevertheless  represent  the  process  diagrammatically,  as  I 
have  done  above,  bearing  in  mind  that  in  the  Wassermann  reaction 
the  factors  designated  as  antigen  and  antibody  are  so  termed  only  for 
sake  of  convenience.  For  the  antibody  in  question  I  would  suggest 
the  term,  lipoidophilic  antibody,  as  denoting  its  essential  characteristic 
and  its  nature  as  a  reaction  product  to  infection. 

PREPARATION  OF  THE  REAGENTS. — 1.  Preparation  of  the  Antigen. 
—While  Wassermann  originally  advocated  the  use  of  saline  extracts 
of  syphilitic  livers,  and  other  investigators  then  showed  that  alco- 
holic extracts  of  normal  organs  (heart,  liver,  kidney)  answer  the 
purpose  as  well,  Noguchi  pointed  out  that  the  "antigenic"  properties 
of  such  extracts  essentially  belong  to  the  acetone-insoluble  fraction, 
and  that  undesirable  "side"  reactions  can  be  avoided  by  utilizing 
this  fraction  only.  For  this  reason  I  have  abandoned  the  use  of 
simple  alcoholic  extracts  altogether,  and  employ  the  acetone-insoluble 
fraction  exclusively.  This  is  prepared  as  follows :  Fifty  or  a  hundred 


DIAGNOSTIC  REACTIONS 


293 


grams  of  beef  heart,  liver,  or  kidney  are  passed  through  a  meat- 
hashing  machine  and  extracted  with  ten  times  the  amount  of  abso- 
lute alcohol  by  standing  for  several  days  at  incubator  temperature. 
The  resultant  mixture  is  filtered  through  ordinary  filter  paper,  the 
filtrate  evaporated  to  dryness  with  the  aid  of  an  electric  fan,  the 
residue  taken  up  with  as  little  ether  as  possible,  and  the  ethereal 
solution  treated  with  five  times  its  volume  of  acetone.  A  precipitate 
forms,  which  is  allowed  to  settle  to  the  bottom,  when  the  supernatant 
fluid  is  poured  off.  From  the  remaining  brown,  sticky  material 
a  saturated  solution  is  prepared  in  absolute  methyl  alcohol,  which 
is  conveniently  put  up  in  glass  beads  or  ampoules,  in  quantities  of 
about  1  c.c.  each. 

Prepared  in  this  manner  the  antigen  keeps  for  many  months 
without  losing  in  strength,  but  should  be  tested  from  time  to  time 
nevertheless.  To  this  end  emulsions  of  varying  strength  are  pre- 
pared with  0.9  per  cent,  saline,  treated  with  constant  amounts  of 
complement,  incubated  for  thirty  minutes  in  a  water-bath  at  37°  to 
40°  C.,  and  then  combined  with  the  hemolytic  system  that  has  been 
chosen  to  ascertain  whether  complement  fixation  has  taken  place 
or  not.  The  general  plan  to  be  followed  is  shown  in  the  accompanying 
table: 


Hemolytic  am- 

Antigen 
dilutions  in 
saline. 

Saline 
0.9% 
solution. 

Guinea-pig     Washed  sheep 
complement       corpuscles 
(linlO).     (5%  emulsion). 

boceptor  (2^ 
times  the  titer 
strength)  .                 Result. 

c.c. 

c.c. 

c.c.                       c.c. 

c.c. 

0.5 

(4.0  in 

10) 

0.5 

0.5 

1 

0.5 

0.5 

A 

No  hemoly- 

—      * 

sis. 

0.5 

(3.0  in 

10) 

0.5 

0.5 

's  • 

0.5 

0.5 

>>H      Partial 

*$ 

.-•^          hemolvsis. 

0.5 

(2.5  in 

10) 

0.5 

0.5 

.s-? 

0.5 

0.5 

Partial 

"S.I 

g  f!          hemolysis. 

0.5 

(2.5  in 

10) 

0.5 

0.5 

(-,  cS 

•2  ^ 

0.5 

0.5 

*"  0> 

Partial 

_,  0) 

o"*3 

hemolysis. 

0.5 

(1.5  in 

10) 

0.5 

0.5 

§3 

0.5 

0.5 

'•^.9 

Complete 

Is 

^    03 

hemolysis. 

0.5 

(1.0  in 

10) 

0.5 

0.5 

0.5 

0.5 

§^ 

Complete 

I 

'1 

hemolysis. 

As  the  antigen  in  itself  is  capable  of  absorbing  a  certain  amount  of 
complement  it  will  be  found  that  with  the  stronger  emulsions  either 
no  hemolysis  at  all  occurs,  or  partial  hemolysis  only  takes  place. 
For  the  actual  experiment  two-thirds  of  that  strength  is  chosen  which 
first  gives  complete  hemolysis.  In  the  above  example  it  will  be 
noted  that  this  was  first  obtained  with  the  1.5  in  10  dilution;  in  this 
case  then  we  would  use  the  antigen  in  a  dilution  of  1  in  10,  and  this 


294  1MMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

would  represent  its  titer.  After  this  has  been  ascertained  the 
antigen  (in  the  dilution  just  determined)  is  next  tested  against 
a  known  syphilitic  serum  and  a  known  normal  serum,  both  having 
been  inactivated  by  heating  for  thirty  minutes  in  the  water-bath 
at  56°  C.,  and  extracted  with  sheep  corpuscles,  as  described  below 
(sub.  5),  0.5  c.c.  of  the  diluted  serum  being  substituted  for  the 
0.5  c.c.  measure  of  saline,  corresponding  to  the  second  column  in  the 
table  above.  With  the  known  syphilitic  serum  no  hemolysis  should 
then  result,  while  with  the  normal  serum  hemolysis  must  be  com- 
plete at  the  expiration  of  thirty  minutes  in  the  water-bath.  Occa- 
sionally it  happens  that  partial  fixation  of  complement  occurs  with 
normal  serum  even.  Such  antigens  are  evidently  not  fit  for  use, 
for  although  every  serum  possesses  anticomplementary  properties 
to  a  certain  extent,  it  would  be  dangerous  to  let  the  boundaries 
of  the  normal  and  the  abnormal  overlap.  In  testing  out  the  antigen 
it  is  further  well  to  set  the  tube,  in  which  complete  fixation  was 
noted  at  the  expiration  of  thirty  minutes,  aside  in  the  ice-box  for  a 
few  hours  and  to  examine  it  from  time  to  time  to  ascertain  whether 
hemolysis  takes  place  on  standing.  If  this  should  be  the  case  to 
any  marked  extent,  the  antigen  probably  possesses  hemolytic 
properties  in  itself  and  is  then  likewise  undesirable.  Formerly  when 
simple  alcoholic  extracts  were  almost  exclusively  in  use  this  was  a  not 
infrequent  occurrence,  but  with  the  extracts  prepared  as  described 
above  it  is  uncommon.  After  the  antigen  has  been  tested  in  these 
various  directions  it  should  be  kept  in  the  dark  and  preferably  in 
the  ice-box.  It  will  then  not  change  its  titer  for  a  number  of  months, 
but  should  not  be  looked  upon  as  a  stable  product.  In  my  labora- 
tory we  test  the  titer  about  once  a  month,  and  have  reason  to  urge 
this  rule  upon  others. 

2.  The  Hemolytic  Amboceptor. — To  prepare  the  hemolytic  ambo- 
ceptor  a  large  rabbit  is  injected  on  two  occasions,  seven  days  apart, 
with  the  washed  corpuscles  corresponding  to  30  c.c.  of  sheeps'  blood, 
which  must  be  obtained  under  aseptic  precautions,  and  after  removal 
of  the  serum  by  centrifugation,  washed  with  at  least  three  changes 
of  sterile  0.9  per  cent,  salt  solution.  Care  should  be  had  each  time, 
after  packing  down  the  corpuscles  by  centrifugation  and  pipetting 
off  the  washings,  to  stir  up  the  corpuscles  in  the  new  portion  of  saline 
that  is  added.  Finally,  the  corpuscles  are  suspended  in  such  an 
amount  of  saline  that  the  volume  injected  equals  that  of  the  full 


DIAGNOSTIC  REACTIONS  295 

blood  which  was  originally  used.  From  nine  to  eleven  days  later, 
according  to  the  amboceptor  content,  which  can  be  readily  ascer- 
tained by  a  preliminary  test  of  a  few  drops  of  blood,  the  animal  is 
bled  to  death,  the  blood  being  collected  under  aseptic  precautions. 
To  this  end  it  is  convenient  to  use  a  test-tube  which  has  been  drawn 
out  into  a  capillary  near  its  closed  end,  at  an  angle  of  about  115 
degrees.  This  is  sealed,  the  open  end  closed  with  cotton,  and  the 
whole  sterilized.  After  the  animal  has  been  anesthetized,  the  neck 
is  shaved,  scrubbed  with  soap  and  alcohol,  and  the  carotid  dissected 
out  through  a  median  incision.  The  tip  of  the  capillary  is  broken 
off  and  the  tube,  moistened  with  sterile  saline,  introduced  into  the 
vessel,  when  the  blood  will  rise  into  the  collecting  tube.  The  capil- 
lary is  quickly  sealed  in  a  flame  and  the  tube  then  placed  on  ice 
for  the  serum  to  separate  out.  Subsequently,  the  serum  is  pipetted 
off  with  a  sterile  pipette,  heated  for  thirty  minutes  at  56°  C.,  treated 
with  carbolic  acid  to  the  extent  of  0.5  per  cent.,  and  may  then  be 
kept  in  a  dark  colored  bottle,  well  corked,  on  ice.  Instead  of  doing 
this  I  find  it  more  convenient  to  fill  small  glass  beads  with  about 
0.5  c.c.  of  the  serum  each,  to  seal  these,  and  to  keep  them  in  an  ice- 
box. The  addition  of  carbolic  acid  is  then  not  necessary. 

The  titer  of  the  amboceptor  should  be  at  least  such  that  0.5  c.c. 
of  a  1  to  2000  dilution  (in  0.9  per  cent,  saline)  will  completely 
hemolyze  0.5  c.c.  of  a  5  per  cent,  emulsion  of  washed  sheep  corpus- 
cles (see  below),  in  the  presence  of  0.5  c.c.  of  a  1  in  10  dilution  of 
guinea-pig  complement  (see  below),  within  thirty  minutes  at  37°  C. 
With  the  two  injections  of  30  c.c.  of  sheep  blood  each,  one  may 
at  times  obtain  a  serum  which  will  still  hemolyze  this  quantity 
of  corpuscles  in  a  dilution  of  1  to  6000.  At  other  times  better  results 
are  obtained  by  giving  the  rabbit  four  or  five  injections  of  5,  10,  15, 
and  20  c.c.  of  washed  corpuscles,  in  succession,  five  days  apart, 
the  animal  being  killed  when  the  desired  titer  has  been  reached. 

Not  every  animal  can  be  brought  to  a  satisfactory  titer,  however, 
and  during  the  winter  months  especially  it  is  not  unusual  to  find 
but  one  animal  of  perhaps  a  dozen  that  will  furnish  a  satisfactory 
amboceptor. 

Using  one  of  the  little  beads  just  mentioned,  I  make  up  a  1  to  100 
stock  dilution  which,  when  kept  on  ice,  will  usually  retain  its  titer 
for  many  weeks,  and  is  used  to  make  up  the  higher  dilutions  on  the 
days  when  these  are  wanted.  It  is  best,  however,  to  test  it  against 


296  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

the  complement  anew  at  least  once  a  week,  as  the  activity  of  the 
complement  varies  considerably  in  different  guinea-pigs.  In  the 
actual  experiment,  viz.,  in  the  study  of  the  patient's  serum,  from  two 
and  one-half  to  three  times  the  completely  hemolyzing  dose  is  used. 

3.  The   Washed  Corpuscles. — The  necessary  amount  of  sheep's 
blood  is  readily  procured  from  a  slaughtering  house.    If  this  is  not 
available,  a  sheep  may  be  kept  near  the  laboratory  and  is  bled  from 
the  ear  as  occasion  demands.     In  the  hemolytic  experiment  it  is 
not  essential  to  work  aseptically.     After  separation  of  the  serum 
the  corpuscles  are  washed  three  times  with  saline,  as  mentioned 
above.  At  last  all  the  fluid  is  carefully  pipetted  off;  from  the  remain- 
ing corpuscles  a  2.5  per  cent,  emulsion  is  prepared  in  saline,  which 
corresponds  to  a  5  per  cent,  emulsion  of  the  native  blood. 

We  use  the  corpuscles  only  on  the  day  on  which  they  are  procured 
and  on  the  one  following.  They  should  be  kept  in  the  ice-box  while 
not  in  use.  If  the  supernatant  fluid  shows  the  least  discoloration 
they  should  be  discarded.1 

4.  The  Complement. — Guinea-pig  serum  is  used  as  complement. 
As  this  is  supposedly  derived  from  disintegrating  leukocytes,  it  is 
recommended  to  obtain  the  blood  some  hours  before  use.     We 
usually  kill  the  guinea-pig  the  evening  before,  by  cutting  the  vessels 
of  the  neck,  after  anesthetizing  the  animal  with  ether.    The  blood 
is  received  in  Petri  dishes  and  is  kept  overnight  on  ice.    The  follow- 
ing morning  the  serum  is  pipetted  off;  if  desired  one  can  then  place 
the  clotted  blood  in  centrifuge  tubes  and  obtain  still  more  serum 
by  centrifugation.    If  it  is  not  practical  to  kill  the  animal  the  evening 
before,  this  may  be  done  in  the  morning  of  the  day  on  which  it  is  used; 
the  blood  is  then  placed  on  ice  for  two  or  three  hours  and  the  serum 
obtained  by  centrifugalizing  the  clot.  Before  use  the  serum  is  diluted 
1  in  10.    The  unused  portion  of  the  concentrated  serum  may  be  kept 
frozen,  for  one  or  two  days,  but  before  further  use  it  must  be  tested 
and  adjusted  to  the  hemolytic  amboceptor  as  described.     Very 
often  it  will  be  found  to  be  inert.    In  my  laboratory,  we  have  set 
aside  special  days  of  the  week  for  complement  fixation  work,  and 
we  then  make  no  attempt  to  preserve  any  of  the  complement. 

Where  only  a  few  specimens  are  to  be  examined  at  one  time  it 

1  For  washing  purposes,  as  well  as  for  diluting  the  various  reagents,  it  is 
essential  to  use  chemically  pure  sodium  chloride.  Some  of  the  tablets  furnished 
by  dealers  will  cause  hemolysis  in  themselves. 


DIAGNOSTIC  REACTIONS  297 

is  not  necessary  to  kill  the  animal.  A  few  cubic  centimeters  of  blood 
can  be  obtained  by  puncturing  the  heart  with  an  antitoxin  syringe, 
under  anesthesia.  My  own  preference,  however,  is  to  kill  the  animal. 

As  has  already  been  indicated,  the  complement,  before  use, 
whether  fresh  or  not,  must  always  be  adjusted  to  the  amboceptor. 

5.  The  Patient's  Serum. — It  is  generally  recommended  to  secure 
blood  from  the  patient  as  well  as  from  the  normal  controls  by  vene- 
puncture.  This,  however,  is  totally  unnecessary.  The  required 
amount  can  be  readily  obtained  from  the  ear.  This  is  punctured 
with  a  small  lancet  or  tenotomy  knife,  introducing  the  blade,  at  an 
angle,  into  the  lobule  and  making  a  small  sweep  of  the  point  of  the 
blade  without  enlarging  the  skin  incision,  so  as  to  cut  a  larger  number 
of  capillaries.  Enough  blood  can  then  be  milked  out  in  about  five 
minutes  to  fill  a  glass  tube  1J  to  2  inches  long,  and  having  an  inside 
diameter  of  J  of  an  inch.  The  tube  is  corked  and  thus  brought 
to  the  laboratory.  The  clot  is  then  separated  from  the  walls  and 
the  corpuscles  packed  down  by  centrifugation.  The  supernatant 
serum  is  pipetted  off  with  Wright  pipettes,  placed  in  tubes  similar 
to  those  in  which  the  blood  is  collected  and  inactivated  (complement 
destruction)  by  heating  for  thirty  minutes  at  56°  C.;  after  this  it  is 
diluted  1  in  5,  and  is  then  ready  for  use.1 

A  normal  serum  and  a  specimen  from  a  known  case  of  syphilis 
should  always  be  available  as  controls. 

It  is  recommended  that  all  sera  should  be  examined  on  the  day  on 
which  they  have  been  procured.  This  no  doubt  is  a  good  rule,  but 
I  have  found  that  fixing  sera  remain  active  for  several  weeks.  It  is 
thus  perfectly  feasible  to  send  specimens  from  a  distance,  especially 
if  the  serum  is  separated  from  the  corpuscles  after  bleeding  the 
patient. 

As  human  serum  frequently  contains  amboceptors  which  are 
hemolytic  for  sheep  corpuscles  in  the  presence  of  complement,  and 
as  their  amount  is  variable  and  at  times  not  inconsiderable,  a  factor 
is  here  introduced  into  the  experiment  which  could  convert  a  positive 
into  a  negative  result.  For  we  must  bear  in  mind  thaf  the  activity 
of  amboceptor  and  complement  stand  in  an  inverse  proportion  to 
one  another,  such  that  a  very  small  amount  of  complement  would 

1  If  it  should  be  desired  to  secure  somewhat  larger  amounts  of  blood  by  vene- 
puncture,  the  vacuum  bulb  recommended  by  Keidel  will  be  found  very  convenient 
(see  Jour.  Amer.  Med.  Assoc.,  May  25,  1912,  p.  1579). 


298  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

be  quite  sufficient  to  effect  a  very  considerable  degree  of  hemolysis, 
if  amboceptor  were  present  in  excess. 

Some  investigators,  such  as  Noguchi,  have  accordingly  recom- 
mended the  use  of  an  antihuman  instead  of  an  antisheep  hemolytic 
system.  I  have  found,  however,  that  this  is  not  only  inconvenient, 
but  also  unnecessary,  as  the  natural  antisheep  amboceptor  can  be 
readily  removed  by  merely  diluting  the  inactivated  serum  of  the 
patient  with  five  times  its  volume  of  the  corpuscle  emulsion,  and 
incubating  the  mixture  for  thirty  minutes  in  the  water-bath  at 
37°  C.;  the  corpuscles  with  the  anchored  "  natural"  antisheep  ambo- 
ceptor are  then  thrown  down  with  the  centrifuge,  when  the  super- 
natant, now  already  diluted,  serum  is  ready  for  use.  This  step  is 
now  carried  out  as  a  matter  of  routine  in  my  own  laboratories,  and 
assures  perfectly  satisfactory  results;  with  the  use  of  the  Noguchi 
antigen,  and  this  modification  the  objectionable  "Nachlosung" 
(continuing  hemolysis  at  the  end  of  the  experiment)  is  no  longer 
a  source  of  error  and  hence  of  worry.  The  test-tubes  which  we  use 
measure  4  inches  in  length,  by  f  inch  inside  diameter. 

METHOD. — When  everything  is  in  readiness  the  complement  and 
amboceptor  are  adjusted  to  one  another,  using  dilutions  of  1  to  1000, 
1  to  2000,  1  to  3000  up  to  1  to  6000  of  the  amboceptor;  0.5  c.c.  is 
our  unit  of  measure,  and  we  accordingly  combine  0.5  of  the  various 
amboceptor  dilutions  with  0.5  c.c.  of  the  complement  (1  in  10)  and 
0.5  c.c.  of  the  corpuscle  emulsion  (5  per  cent.).  The  tubes  are  placed 
in  the  water-bath  at  37°  C.,  and  are  frequently  shaken.  At  the 
expiration  of  thirty  minutes  the  highest  dilution  is  noted  at  which 
complete  hemolysis  occurs.  The  amboceptor  dilution  to  be  used  in 
the  actual  experiment  is  then  made  2J  to  3  times  as  strong.  Thus, 
if  complete  hemolysis  occurred  at  1  to  6000,  we  would  use  a  1  to 
2500  or  a  1  to  2000  dilution. 

The  antigen  has  been  previously  tested,  as  described.  With 
human  heart  antigen,  one  can  usually  use  a  dilution  of  1  to  10. 

The  titers  of  the  various  reagents  having  thus  been  ascertained, 
the  experiment  proper  can  now  be  carried  out  (tubes  marked  E), 
using  0.5  c.c.  of  the  patient's  serum  (1  in  5)1  combined  with  0.5  c.c. 

1  The  patient's  serum  has  previously  been  freed  of  any  natural  antisheep 
amboceptors  by  diluting  it  (1  in  5)  with  the  standard  emulsion  of  sheep  cor- 
puscles and  incubating  for  thirty  minutes,  after  which  the  corpuscles  are  thrown 
down  by  centrifugation,  when  the  supernatant  fluid  is  pipetted  off  and  can  be 
immediately  used  in  the  experiment. 


PLATE   VI 


Wassermann  Reaction. 

A,  positive;  B,  partial;   C,  negative  reaction. 

Note  undissolved  blood  corpuscles  in  .-1,  partial  hemolysis  in  B,  and  complete  hemolysia  in  C. 


DIAGNOSTIC  REACTIONS  299 

of  complement  (1  in  10)  and  0.5  c.c.  of  antigen  (1  in  10).  At  the 
same  time  controls  (tubes  marked  C)  are  prepared  in  which  the 
antigen  is  left  out,  so  that  0.5  of  each  serum  is  combined  with  0.5  c.c. 
of  complement  and  0.5  c.c.  of  saline  (in  place  of  the  antigen).  The 
E  and  C  tubes  properly  marked  with  the  patient's  numbers  are 
placed  in  the  water-bath  for  thirty  minutes  and  then  receive,  each, 
0.5  c.c.  of  the  hemolytic  amboceptor  and  0.5  c.c.  of  the  corpuscles. 
They  are  then  returned  and  left  for  thirty  minutes  longer,  the  tubes 
being  frequently  shaken.  After  that  some  writers  recommend  that 
they  be  placed  on  ice  and  examined  the  next  morning.  I  can  see 
no  advantage  in  this  delay,  and  prefer  to  centrifugalize  the  tubes 
and  read  them  at  once.  Strictly  speaking  it  is  not  necessary  to  wait 
even  thirty  minutes  if  one  places  a  tube  containing  antigen-comple- 
ment-normal  serum  in  the  lot  and  breaks  off  the  incubation  as  soon 
as  this  control  is  completely  hemolyzed. 

RESULTS. — Complete  inhibition  or  absolute  fixation  is,  of  course, 
at  once  evident  from  the  fact  that  the  supernatant  fluid  (after  centri- 
fugation)  is  perfectly  colorless,  the  corpuscles  being  all  at  the  bottom. 
Partial  fixation  will  show  itself  by  a  more  or  less  colored  supernatant 
fluid  and  the  presence  of  a  varying  number  of  undissolved  red  cells 
at  the  bottom,  while  with  complete  hemolysis  there  is  no  sediment 
of  red  cells  whatever.  The  results  are  accordingly  noted  as  +  ++, 
++,  +,  ±,  and  0  (see  Plate  VI). 

On  the  question  of  a  well-marked  fixation  there  can,  of  course, 
be  no  dispute,  but  with  slight  fixations  errors  are  very  apt  to 
creep  in.  We  would  suggest  that  slight  fixation  be  neglected  and 
reexaminations  made,  especially  in  cases  which  are  submitted  for 
diagnosis. 

The  controls  will  usually  be  found  hemolyzed  completely,  but  at 
times,  though  rarely,  sera  are  met  with  which  fix  more  or  less  com- 
pletely by  themselves.  In  such  cases  it  would,  of  course,  not  be 
warrantable  to  say  that  the  reaction  in  the  E  tube  was  due  to 
syphilis.  What  this  independent  inhibition  means  we  do  not  know. 

Regarding  the  value  of  the  Wassermann  reaction,  both  from  the 
standpoint  of  diagnosis  and  in  its  bearing  upon  the  question  of 
treatment,  we  would  emphasize  that  its  neglect  in  a  doubtful  case 
from  either  point  of  view  would  constitute  a  grave  Kunstfehler,1  as 

1  An  error  of  omission  would  approximately  express  the  idea  in  our  own 
language. 


300  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

the  Germans  put  it,  of  which,  very  fortunately,  but  few  modern 
physicians  are  apt  to  be  guilty. 

Considered  from  the  diagnostic  standpoint  a  well-pronounced 
positive  reaction  may  probably  always  be  regarded  as  indicating  the 
existence  of  syphilis,  if  we  can  rule  out  such  diseases  as  frambesia, 
leprosy,  sleeping  sickness,  and  scarlatina.  In  malignant  disease  a 
certain  degree  of  complement  fixation  may  also  be  obtained,  in  a 
considerable  number  of  cases,  but  I  have  not  been  able  to  convince 
myself  that  a  triple  plus  (+  ++)  reaction  can  ever  be  ascribed  to 
the  malignant  process  in  itself.  Partial  reactions  (+  or  ±),  on  the 
other  hand,  are  here  not  infrequently  met  with  and  may  even  be 
seen  in  persons  who  neither  show  any  present  signs  nor  give  any 
history  of  syphilis  in  the  past.  What  these  feeble  reactions  mean 
we  do  not  know,  but  we  are  inclined  to  think  that  they  may  possibly 
be  the  expression  of  some  inherited  syphilitic  taint,  though  we  have 
but  few  data  to  support  this  belief.  As  the  result  of  a  fairly  wide 
experience  with  the  reaction  we  have  come  to  the  conclusion  that 
from  the  diagnostic  standpoint  triple  plus  reactions  only  should  be 
considered  as  positive  evidence  of  syphilis,  while  the  feebler  grades,  in 
individuals  with  an  admitted  history  of  the  disease,  may  be  regarded 
as  indicating  that  the  infection  is  probably  limited  to  relatively 
small  areas,  from  which  an  insignificant  absorption  of  spirochetal 
substance  is  taking  place,  with  a  correspondingly  limited  formation 
of  the  lipoidophilic  antibody.  If  the  actual  focus  of  infection  should 
be  sufficiently  restricted  it  is,  of  course,  conceivable  that  a  negative 
reaction  even  might  be  obtained,  and  it  is  for  this  reason  that  a 
single  negative  reaction  has  a  limited  value  only  from  the  standpoint 
of  diagnosis  as  well  as  of  treatment.  As  pointed  out  in  a  pre- 
vious chapter,  however  (see  section  on  Salvarsan),  it  is  frequently 
possible  by  the  administration  of  a  few  large  doses  of  mercury  to 
evoke  a  positive  reaction  in  individuals  in  whom  the  disease  has 
almost  been  eradicated,  whereby  a  larger  number  of  spirochetes  is 
destroyed  at  one  time  and  a  more  intense  stimulus  given  to  antibody 
formation  (provocatory  stimulation).  This  possibility  has  not  yet 
received  the  recognition  which  it  deserves,  but  should  be  utilized  in 
all  doubtful  cases,  as  well  as  in  determining  whether  a  continuance 
of  treatment  is  desirable  or  not  (see  page  274). 

In  very  early  cases  of  syphilis,  in  which  a  sufficient  length  of  time 
for  the  formation  of  antibodies  has  not  yet  elapsed,  the  result  will, 


DIAGNOSTIC  REACTIONS 


301 


of  course,  also  be  negative,  but  in  these  the  diagnosis  can  usually  be 
made  by  direct  demonstration  of  the  spirochete  with  the  microscope. 
With  the  limitations  just  set  forth  a  diagnosis  of  syphilis  can  be 
reached  by  means  of  the  Wassermann  reaction  in  over  90  per  cent, 
of  the  cases  taken  at  random,  the  different  types  giving  different 
values,  as  shown  in  the  accompanying  table,  which  is  taken  from 
Noguchi.  The  values  given  were  obtained  with  the  Noguchi  system, 
i.  e.,  with  an  antihuman  hemolytic  system,  but  represent  practically 
what  the  method  furnishes  which  we  have  described  above. 


NOGUCHI  SYSTEM;  SYPHILIS,  PARASYPHILIS,  HEREDITARY  SYPHILIS,  AND 
SYPHILIS  SUSPECTS 


Primary  syphilis 
Secondary  syphilis 
Tertiary  syphilis 
Early  latent  syphilis 
Late  latent  syphilis 
Under  prolonged  treatment 
Cerebral  syphilis 

Tabes 

General  paralysis 
Hereditary  syphilis 
Syphilis          


.1 

2)  fl 

of 

V 

+ 

- 

± 

No. 

Per  cent. 

70 

65 

92.8 

4 

1 

197 

190 

96.0 

5 

2 

177 

159 

89.9 

16 

2 

115 

87 

75.6 

24 

4 

150 

119 

79.3 

27 

4 

39 

4 

10.2 

32 

3 

5 

3 

60.0 

1 

1 

125 

85 

68.0 

27 

13 

15 

13 

86.0 

2 

0 

17 

17 

100.0 

0 

0 

172 

60 

34.8 

96 

16 

1082 

802 

234 

46 

COMPARISON  OF  THE  WASSERMANN  AND  NOGUCHI  SYSTEMS 


Cases 
examined 

Wassermann 

Noguchi 

+ 

+ 

No. 

Per  ct. 

No. 

Per  ct. 

Primary  syphilis  .... 

23 

17 

73.9 

6 

20 

86.9 

3 

Secondary  syphilis 

79 

69 

87.3 

10 

76 

96.2 

3 

Hereditary  syphilis    . 

65 

52 

80.0 

13 

57 

87.6 

8 

Early  latent  syphilis 

27 

13 

48.0 

14 

18 

66.6 

9 

Late  latent  syphilis    . 

32 

24 

75.0 

8 

27 

84.3 

5 

Tabes  

18 

8 

44.0 

10 

13 

72.2 

5 

244 

183 

61 

211 

33 

302  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

COMPARISON  OF  THE  WASSERMANN  AND  NOGUCHI  SYSTEMS  (RESULTS 
OBTAINED  BY  D.  M.  KAPLAN) 


x 

9 

Wassermann 

Noguchi 

+ 

+ 

No. 

Per  ct. 

No. 

Per  ct. 

Primary  syphilis     .... 

138 

122 

90 

16 

134 

97 

4 

Secondary  syphilis 

281 

242 

86 

39 

270 

98 

11 

Tertiary  syphilis     .... 

191 

140 

73 

51 

155 

81 

36 

Latent  syphilis       .... 

79 

41 

51 

38 

60 

75 

19 

Hereditary  syphilis 

20 

18 

90 

2 

18 

90 

2 

Tabes     

205 

125 

60 

80 

134 

65 

71 

General  paresis       .... 

61 

40 

65 

21 

44 

72 

17 

Cases  for  diagnosis 

311 

98 

31 

213 

180 

57 

131 

1286 

826 

460 

995 

291 

As  regards  the  relation  of  the  Wassermann  reaction  to  the  treat- 
ment of  syphilis  with  salvarsan  or  salvarsan  in  combination  with 
mercury,  the  majority  of  syphilographers  are  in  accord  in  demanding 
that  the  treatment  be  continued  until  a  permanently  negative  Wasser- 
mann is  obtained  and  maintained  (see  section  on  Salvarsan).  This 
standpoint  is  in  accord  with  the  view  that  the  Wassermann  reaction 
is  a  reaction  of  infection  and  not  of  immunity,  and  that  the  existence 
of  infection  may  be  inferred  so  long  as  the  reaction  is  demonstrable. 

The  rapidity  with  which  the  reaction  disappears  under  treatment 
is  quite  variable.  I  have  thus  obtained  a  persistingly  negative  result 
already  after  a  single  injection  of  salvarsan,  while  in  other  cases  the 
salvarsan  in  itself,  though  given  repeatedly,  was  not  able  to  cause 
the  reaction  to  disappear,  whereas  this  promptly  occurred,  if  mer- 
curial treatment  was  instituted  in  addition.  For  further  details  of 
this  order,  however,  I  must  refer  the  reader  to  special  works. 

In  this  connection  it  is  interesting  to  note  that  Noguchi  has 
recently  compared  the  findings  obtained  with  the  Wassermann 
technique,  i.  e.,  with  the  use  of  lipoid  antigen,  with  the  results 
which  were  obtained,  when  a  pure  culture  of  spirochetes  was  used 
as  antigen.  I  append  some  of  the  more  important  conclusions  to 
which  these  investigations  gave  rise:  "(1)  The  Wassermann  reac- 
tion is  caused  by  lipotropic  substances,  but  not  by  the  antibodies 
which  combine  specifically  with  the  pallida  antigen;  (2)  the  fixation 
produced  by  the  culture  pallida  antigen  with  certain  syphilitic  sera 
is  caused  by  the  specific  antibodies  contained  in  the  latter  and  may 
constitute  a  specific  diagnostic  method  for  syphilis;  (3)  the  fixation 


DIAGNOSTIC  REACTIONS  303 

caused  by  a  syphilitic  testicular  extract  behaves  like  the  culture 
pallida  extract  in  the  majority  of  cases,  but  when  the  sera  (syphilitic 
or  leprous)  contain  abundant  lipotropic  substances,  it  may  give  a 
Wassermann  reaction  as  well,  which  is  not  the  case  with  the  culture 
pallida  antigen;  and,  finally,  (4)  in  the  serum  of  rabbits  with  active 
syphilitic  orchitis  there  is  no  indication  of  the  presence  of  a  suffi- 
cient amount  of  the  antibodies  for  the  pallida  antigen,  although 
it  gives  a  strong  Wassermann  reaction.  It  remains  to  be  seen  when 
and  under  what  conditions  the  specific  antibodies  for  the  pallida 
will  most  abundantly  be  formed  in  syphilitic  patients.  At  all  events 
it  is  rather  remarkable  that  the  amount  of  the  antibodies  detectable 
by  the  pallida  antigen  in  these  cases  was  so  small  as  compared  with 
certain  other  infectious  diseases,  in  this  respect.  It  is  not  improb- 
able that  those  who  come  under  our  care  belong  to  a  class  of  indi- 
viduals with  comparatively  less  resistance  to  the  pallida  and  are 
incapable  of  producing  sufficient  antibodies,  while  there  are  many 
who  respond  to  the  infection  with  more  vigorous  formation  of  the 
antibodies  and  reduce  the  infection  to  a  harmless  latency  or  even 
destroy  the  pallida  completely.  This  latter  class  of  infected  persons 
do  not,  of  course,  frequent  our  clinics.  If  this  is  the  case,  it  would 
be  of  immense  prognostic  importance  to  check  a  patient  from  the 
beginning  of  infection  by  the  complement  fixation  test  with  the 
pallida  antigen,  thereby  determining  the  resistance  of  the  patient 
against  the  disease. 

"We  have  in  the  Wassermann  reaction  a  fair  measure  of  activity 
of  the  infecting  agent,  and  now  we  will  have  in  the  pallida  fixation 
reaction  a  gauge  for  the  defensive  activity  of  the  infected  host." 

While  the  principle  of  complement  fixation  has  thus  far  found 
its  widest  field  of  practical  application  in  the  diagnosis  of  syphilis, 
in  the  form  of  the  Wassermann  reaction,  as  just  described,  there  is 
reason  for  thinking  that  the  diagnosis  of  latent  gonococcus  infections 
also  will  ere  long  be  possible  upon  the  same  experimental  lines. 
Excellent  results  have  already  been  reported  from  different  sources. 

Aside  from  this  possibility  the  same  principle  may  also  be  utilized 
in  legal  medicine  when  the  question  arises  whether  a  certain  blood- 
stain is  of  human  origin  or  not.  In  such  a  case  the  material  in 
question  is  brought  into  solution  and  is  then  tested  as  antigen  against 
an  active  antihuman  precipitating  serum  (see  Precipitin  Reaction, 
below),  which  has  been  obtained  by  immunizing  rabbits  with  human 


304  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

blood  serum,  this  antiserum  taking  the  place  of  the  antibody  of  the 
Wassermann  reaction.  If,  then,  the  suspected  substance  contains 
human  albumins  these  will  react  with  the  corresponding  precipitin 
of  the  antiserum,  with  the  result  that  any  complement  that  may 
simultaneously  be  present  is  bound  to  a  greater  or  less  extent  exactly 
as  in  the  case  of  the  Wassermann  reaction. 


PRECIPITIN   REACTIONS 

Following  the  demonstration  by  Tchistovitch  and  Bordet  (1899) 
that  not  only  vegetable  albumins  but  animal  albumins  also  are 
capable  of  giving  rise  to  precipitin  formation,  when  injected  into 
animals  of  an  alien  species,  Uhlenhuth  especially  drew  attention 
to  the  remarkable  specificity  of  the  reaction  when  applied  to  the 
study  of  the  blood  of  different  animals.  He  thus  laid  the  founda- 
tion of  the  modern  biological  blood  test,  which  is  now  recognized  as 
proper  evidence  regarding  the  origin  of  blood-stains,  in  the  courts 
of  practically  all  civilized  countries. 

Aside  from  these  more  practical  bearings  the  precipitin  reaction 
has  attracted  a  great  deal  of  attention  owing  to  the  unexpected 
light  which  it  has  thrown  upon  the  biological  relationship  existing 
between  different  animals.  For  it  has  been  shown  that  while  the 
precipitins  which  can  be  produced  in  a  rabbit,  for  example,  by  the 
injection  of  the  serum  of  a  horse  and  which  naturally  wTill  react 
with  the  latter,  likewise  do  so  with  the  serum  of  the  donkey  and 
the  tapir.  An  antidog  serum  will  similarly  react  with  the  serum  of 
the  fox,  antichicken  serum  with  pigeon  serum,  antigoat  serum  with 
sheep  and  bovine  serum,  antihuman  serum  with  the  serum  of 
apes,  etc. 

These  group  reactions  are  readily  explained  if  we  assume  the 
existence  in  the  antigenic  sera  of  "partial"  precipitinogens,  i.  e., 
of  precipitinogenic  molecular  complexes  which  are  peculiar  to  a 
special  species,  besides  others  which  are  common  to  a  whole  group 
of  species,  all  of  which  will  naturally  give  rise  to  corresponding 
"partial"  precipitins,  in  a  manner  quite  analogous  to  the  formation  of 
"partial"  agglutinins  (which  see).  That  such  partial  precipitins 
actually  exist  in  an  antiserum  may  be  shown  by  treating  antihuman 
serum  with  monkey  serum  when  the  antimonkey  precipitin  will 


PRECIPITIN  REACTIONS  305 

cause  the  formation  of  a  corresponding  precipitate.  If  this  is  then 
removed  by  centrifugation  (quantitative  relations  being,  of  course, 
duly  considered)  the  remaining  serum  may  be  shown  to  have  retained 
its  precipitin  for  human  serum,  while  that  for  monkey  serum  has 
disappeared.  That  antihuman  serum,  moreover,  should  possess  a 
larger  quantity  of  antihuman  than  of  antimonkey  precipitin  would 
naturally  suggest  itself  and  can  be  demonstrated  by  suitable  methods. 

The  technique  which  is  involved  in  these  various  examinations 
may  be  suitably  described  in  connection  with  its  application  to  the 
medico-legal  blood  test,  according  to  Uhlenhuth. 

The  Biological  Blood  Test. — As  the  legal  question  at  issue  is  usually 
whether  or  not  a  certain  blood-stain  is  of  human  origin,  it  is  ordinarily 
only  necessary  to  examine  the  material  in  question  in  reference  to 
its  behavior  toward  an  antihuman  serum.  If,  on  the  other  hand, 
the  antihuman  investigation  has  shown  that  the  material  was  not 
of  human  origin,  and  it  is  desired  to  ascertain  from  what  animal 
species  the  blood  was  derived,  corresponding  sera  must,  of  course, 
also  be  available. 

PREPARATION  OF  THE  ANTISERA. — The  antisera  in  question  are 
usually  obtained  from  rabbits  after  injection  with  either  human 
serum,  pig  serum,  or  bovine  serum,  etc.,  as  the  case  may  be.  The 
injections  are  given  intraperitoneally  or  intravenously  at  intervals 
of  five  or  six  days,  using  10,  8,  and  5  c.c.  respectively  in  the  first 
instance,  and  5,  3,  and  2  c.c.  if  the  latter  method  is  preferred.  It  is 
always  best  to  inject  several  rabbits  at  the  same  time,  especially  since 
not  every  animal  furnishes  a  serum  with  a  sufficiently  high  titer. 
According  to  Uhlenhuth  this  should  be  such  that  0.1  c.c.  of  the  anti- 
serum  shall  produce  a  distinct  turbidity  either  instantaneously  or  at 
most  after  one  to  two  minutes,  when  added  to  1  c.c.  of  a  1  to  1000 
dilution  of  the  corresponding  antigenic  serum.  Added  to  1  c.c.  of  a 
1  to  10,000  and  1  to  20,000  dilution  a  turbidity  should  be  discernible 
after  three  and  five  minutes  respectively,  while  a  control  specimen, 
containing  only  0.85  per  cent,  saline  (the  diluent  in  question)  and 
0.1  c.c.  of  the  antiserum  must,  of  course,  remain  clear.  The  reaction 
is  best  observed  by  holding  the  tubes  (without  shaking)  against  a 
dark  background,  when  it  will  be  seen  that  the  turbidity  first  appears 
as  a  faint  opalescence  at  the  bottom  of  the  tubes,  but  in  the  course 
of  five  minutes  extends  throughout  the  specimen,  becoming  increas- 
ingly denser  and  ultimately  settling  to  the  bottom  as  a  precipitate. 
20 


306  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

The  desired  titer  is  frequently  obtained  already  on  the  sixth  day 
following  the  last  injection.  If  an  examination  of  a  test  specimen 
taken  from  the  ear  does  not  indicate  the  desired  strength  at  this 
time,  it  may  be  necessary  to  give  a  fourth,  a  fifth,  and  even  a  sixth 
injection,  but  it  may  also  happen  that  the  particular  animal  cannot 
be  brought  to  the  titer  that  is  necessary,  with  any  number  of  injec- 
tions. If,  however,  the  examination  shows  that  the  serum  can  be 
used,  the  animal  is  bled  to  death,  the  serum  separated  by  centrifu- 
gation,  cleared  by  passing  through  a  Berkefeld  filter,  and  finally 
stored  in  little  glass  beads  or  ampoules  in  portions  of  1  c.c.  each. 
No  preservative  is  added,  and  it  is  accordingly  necessary  throughout 
to  observe  aseptic  precautions. 

All  examinations  are  conducted  in  little  test-tubes,  such  as  those 
used  in  the  Wassermann  work,  which  must,  of  course,  be  scrupulously 
clean.  Or,  if  very  small  amounts  of  material  only  are  available 
for  the  examination,  this  is  conducted  in  glass  capillaries.  The 
turbidity  then  develops  at  the  zone  of  contact  between  the  two 
fluids,  and  may  be  advantageously  observed  with  the  aid  of  a 
magnifying  glass. 

PREPARATION  OF  THE  SUSPECTED  MATERIAL. — This  is  brought 
into  solution  with  the  aid  of  0.85  per  cent,  saline,  and  is  then  further 
diluted  to  such  a  degree  that  on  boiling  a  small  amount  (1  c.c.)  with 
a  drop  of  25  per  cent,  nitric  acid  a  slight  opalescence  develops.  This 
would  correspond  to  a  1  to  1000  dilution  of  the  blood  in  its  original 
state  and  represents  the  minimal  degree  of  dilution  (i.  e.,  the  maximal 
concentration)  with  which  the  actual  test  should  be  made. 

The  solution  of  the  suspected  material  should,  of  course,  also 
be  perfectly  clear,  to  which  end  it  may  be  necessary  to  pass  it 
through  a  Berkefeld  filter,  or,  if  the  quantity  be  small,  through  a 
Silberschmidt  microfilter. 

THE  EXAMINATION  PROPER. — Six  tubes  are  placed  in  a  suitable 
rack  and  labelled  I,  II,  III,  IV,  V,  and  VI.  Tube  I  and  II  receive 
1  c.c.  of  the  solution  under  investigation,  III  and  IV  1  c.c.  of  two 
control  solutions  made  up  from  dried  animal  blood,  i.  e.,  from  blood 
which  does  not  correspond  to  the  antiserum  that  is  used,  e.  g.,  cat  or 
dog  blood,  if  the  antiserum  is  antihuman  in  character,  and  which  has 
likewise  been  diluted  so  as  to  correspond  to  a  1  to  1000  solution  (see 
preceding  section),  V  1  c.c.  of  sterile  0.85  per  cent,  saline,  and  VI 
1  c.c.  of  a  1  to  1000  solution  of  blood  (made  up  of  dried  material) 


PLATE   VII 


Cutaneous  Tuberculin   Reaction  of  v.   Pirquet. 
(Taken  from   Hamill.) 


FERMENT  REACTIONS  307 

corresponding  to  the  antiserum  in  question,  i.  e.,  of  human  blood,  if 
the  antiserum  was  antihuman  in  character.  To  each  tube,  with  the 
exception  of  tube  II  (which  is  treated  with  0.1  c.c.  of  normal  rabbit 
serum),  0.1  c.c.  of  the  corresponding  antiserum  is  then  added  in 
such  a  manner  that  the  serum  flows  down  the  side  of  the  tube  and 
does  not  drop  directly  into  the  fluid  below.  The  tubes  are  now 
allowed  to  stand  at  room  temperature  and  without  shaking  for 
twenty  minutes,  when  the  final  reading  is  made.  If  the  result  is 
positive,  i.  e.,  if  the  suspected  material  was  of  human  origin,  precipi- 
tation will  occur  in  tubes  I  and  VI,  while  II,  III,  IV,  and  V  remain 
clear. 

With  this  method  reliable  results  can  be  obtained,  so  long  as  the 
material  under  examination  contains  albumins  which  are  still  cap- 
able of  undergoing  solution,  even  though  they  be  present  only  in 
traces.  Uhlenhuth  and  Beumer  thus  mention  that  they  obtained 
positive  results  with  blood  which  had  undergone  putrefaction  and 
had  been  left  exposed  to  the  air  for  two  years,  as  well  as  with  dried 
blood-stains  which  were  more  than  fifty  years  old. 

FERMENT   REACTIONS 

The  Pregnancy  Test  of  Abderhalden. — This  test  is  primarily  based 
upon  the  observation  that  the  parenteral  introduction  of  complex 
foodstuffs  of  alien  origin  either  leads  to  the  appearance  in  the  blood 
serum  of  reaction  products  de  now  which  are  capable  of  causing  the 
change  of  such  bodies,  or  it  increases  the  quantity  of  corresponding 
products  which  may  normally  be  present.  Rabbit  serum  has  thus 
no  digestive  properties  for  silk  peptone,  while  the  serum  of  an  animal 
that  has  been  previously  injected  with  such  material  is  capable  of 
effecting  its  cleavage.  Similarly  we  find  that  normal  serum  is  incap- 
able of  bringing  about  the  cleavage  of  cane  sugar,  while  the  serum 
of  an  animal  that  has  previously  been  injected  with  the  carbo- 
hydrate in  question  does  this  quite  readily.  Most  extensive  investi- 
gations along  these  lines  convinced  Abderhalden  that  such  a  reaction 
on  the  part  of  the  treated  animal  is  of  invariable  occurrence,  and  in  a 
general  way  this  corresponds  to  our  views  regarding  antibody  forma- 
tion, as  set  forth  in  the  first  part  of  this  volume.  Without  entering 
into  the  question  regarding  the  possible  identity  of  the  antibodies 
in  the  sense  of  Ehrlich  and  the  digestive  reaction  products  with 


308  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

which  we  have  just  become  acquainted,  the  thought  naturally  sug- 
gests itself  that  any  component  of  the  body  may  in  the  end  be  viewed 
as  alien  to  other  parts  of  the  body,  if  it  is  placed  in  surroundings 
which  are  in  reality  alien  to  that  particular  component.  It  is  thus 
quite  conceivable  that  the  presence  in  the  circulation  of  some  of 
the  body's  own  cells  might  give  rise  to  similar  reactions  providing 
that  the  cells  in  question  are  really  foreign  to  the  interior  of  the 
bloodvessels,  i.  e.,  to  the  blood  plasma  or  the  blood  cells.  As  this 
takes  place  not  only  under  various  pathological  conditions,  but  even 
during  pregnancy,  where  chorion  cells  have  been  shown  to  enter 
the  circulation,  it  was  natural  to  put  the  question  to  the  experimental 
test.  As  a  result  Abderhalden  announced  that  he  succeeded  in 
demonstrating  that  the  blood  serum  of  pregnant  animals  has 
acquired  the  power  of  causing  the  cleavage  of  placental  peptone 
and  placental  proteins. 

While  his  experiments  were  originally  conducted  by  bringing 
together  the  serum  of  the  pregnant  animal  and  the  placental  peptone 
in  the  tube  of  the  polarimeter,  when  a  gradual  change  in  the  degree 
of  rotation  could  be  noted  as  the  cleavage  took  place,  he  subsequently 
discovered  that  the  same  reaction  can  be  demonstrated  by  placing 
the  placental  tissue  together  with  the  blood  serum  of  the  individual 
in  a  dialyzing  tube  and  then  testing  the  dialyzate  for  biuret.  To 
this  end  the  following  procedure  is  recommended. 

Abderhalden's  Test. — Preparation  of  the  Antigen. — A  fresh  placenta 
is  stripped  of  its  membrane,  cut  into  small  pieces  and  washed  free 
from  blood  by  kneading  the  material  in  running  water.  It  is  then 
placed  in  boiling  water  which  has  been  slightly  acidified  with  acetic 
acid  (2  drops  of  the  glacial  acid  to  a  liter  of  water),  and  the  boiling 
continued  for  five  minutes.  The  supernatant  fluid  is  decanted  and 
replaced  by  a  corresponding  quantity  of  water.  The  boiling  is  con- 
tinued for  five  minutes  longer,  and  the  water  then  tested  for  biuret 
(see  below).  So  long  as  a  positive  reaction  is  obtained  the  tissue 
is  boiled  with  new  portions  of  water,  until  finally  it  is  placed  in  a 
bottle  (together  with  the  last  portion  of  water)  and  covered  with 
a  layer  of  toluol.  In  this  form  it  keeps  practically  indefinitely.  It 
is  advisable  nevertheless  to  assure  one's  self  from  time  to  time  that 
the  biuret  reaction  is  still  negative;  if  this  should  not  be  the  case 
the  material  is  boiled  with  new  portions  of  water  until  the  desired 
end  has  been  attained. 


PLATE  VIII 


Cutaneous  Tuberculin  Reaction  of  More. 
(Taken   from    Hamill.) 


PLATE  IX 


Tuberculin  Ophthalmo-reaetion.     (Taken  from  Citron.) 


FERMENT  REACTIONS  309 

Diffusion  Tubes. — For  purposes  of  dialysis  Abderhalden  recom- 
mends parchment  tubes  prepared  by  Schleicher  and  Schiill  (No.  597). 
These  may  be  kept  in  water  covered  with  toluol  and  should  never 
be  used  while  dry  or  without  being  previously  tested  for  their 
tightness.  To  this  end  a  small  quantity  of  normal  serum  is  dialyzed 
under  toluol  against  distilled  water  (as  described  below)  for  twenty- 
four  hours,  when  the  dialyzate  is  tested  for  biuret.  Should  the 
reaction  be  positive  it  is  clear  that  the  tube  is  not  tight  enough. 
Judd  found  in  my  laboratory  that  there  are  only  about  six  tubes  in 
twenty-five  (of  the  number  which  Abderhalden  recommends)  which 
are  serviceable  in  this  respect,  the  majority  permitting  the  diffusion 
of  proteins  w^hich  will  likewise  react  with  the  new  triketo  reagent, 
which  is  usually  employed  in  testing  for  biuret.  On  the  other  hand 
it  is  well  to  test  the  tubes  with  a  little  Witte  peptone  solution,  so  as 
to  make  sure  that  they  are  not  too  tight.  This,  however,  seems  to  be 
rare. 

After  use  the  tubes  should  be  washed  in  running  water,  until  the 
surrounding  liquid  no  longer  gives  the  biuret  reaction.  They  are 
then  kept  in  water  under  toluol  until  they  are  needed  again,  or,  as 
Judd  recommends,  they  may  be  suspended  in  distilled  water  in  the 
corresponding  cylinders,  the  latter  stoppered  with  cotton,  and  steril- 
ized by  steam,  when  they  are  ready  for  use. 

The  Biuret  Test. — While  fairly  satisfactory  results  may  be  obtained 
with  the  old  copper  sulphate  test,  usin^  5  c.c.  of  33  per  cent, 
caustic  soda  solution  for  10  c.c.  of  the  dialyzate,  mixing  well 
and  superimposing  a  layer  measuring  0.25  to  0.5  cm.  of  an  0.25 
per  cent,  solution  of  copper  sulphate,  the  triketohydrinden  hydrate 
(Ninhydrin)  test  is  in  many  respects  to  be  preferred  and  much  more 
sensitive.  For  this  reason  it  would  not  be  advisable  to  use  the  latter 
test  in  the  final  examination  if  the  less  sensitive  copper  sulphate 
reaction  only  has  been  used  in  testing  the  placental  tissue  for 
biuret.  The  triketo  reagent  is  a  white  crystalline  substance,  which 
is  employed  in  a  1  per  cent,  aqueous  solution.  On  adding  0.2  c.c. 
of  this  to  a  solution  of  peptone  a  violet  color  develops  on  boiling. 
(This  may  be  tried  with  a  very  dilute  solution  of  Witte  peptone.) 

Preparation  of  the  Serum. — The  blood  should  be  obtained  under 
aseptic  precautions  and  examined  as  fresh  as  possible.  From  5  to 
10  c.c.  are  best  withdrawn  with  a  syringe  from  one  of  the  veins  at 
the  bend  of  the  elbow.  The  serum  is  separated  from  the  corpuscles 
by  centrifugation  in  the  usual  manner. 


310  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

The  Test  Proper. — From  0.5  to  1  gram  of  the  placental  tissue  to- 
gether with  2  or  3  c.c.  of  the  serum  is  placed  in  one  of  the  diffusion 
tubes  (A)  that  has  previously  been  adjusted  in  a  small  cylinder 
containing  about  15  to  20  c.c.  of  distilled  water,  and  both  the  serum 
and  the  outer  fluid  covered  with  a  layer  of  toluol.  A  second  tube 
(B)  is  similarly  arranged,  but  receives  serum  only,  and  no  placental 
tissue,  while  in  a  third  tube  (C)  placental  tissue  is  placed  together 
with  2  or  3  c.c.  of  the  water  in  which  it  was  kept,  all  the  tubes  and 
cylinders  being  guarded  against  putrefactive  changes  by  the  addition 
of  a  liberal  quantity  of  toluol.  The  three  cylinders  are  then  placed 
in  the  incubator  and  left  for  twelve  to  twenty-four  hours,  when  the 
dialyzate  (10  c.c.)  is  tested  for  biuret  as  described.  If  the  test  is 
satisfactorily  positive  tube  B  and  C  should  give  a  negative  reaction 
and  A  a  positive  reaction. 

Results. — Abderhalden  claims  that  a  positive  reaction  may  be 
obtained  in  every  case  of  pregnancy  even  as  early  as  the  first  month 
and  that  the  reaction  persists  until  about  the  end  of  the  first  week 
of  the  puerperal  period.  Corresponding  results  were  reached  in 
pregnant  cows,  dogs,  guinea-pigs,  and  rabbits  (when  a  homologous 
placental  antigen  must  of  course  be  used) .  Other  observers  who  have 
repeated  Abderhalden' s  work  have  obtained  corresponding  results 
and  it  would  seem  that  the  method  may  be  regarded  as  one  whose 
value  is  already  beyond  dispute. 

That  the  same  principle  may  be  of  use  in  the  diagnosis  of  patho- 
logical conditions  also  seems  quite  possible  and  Abderhalden  himself 
has  already  pointed  out  that  cancer  would  suggest  itself  as  a  profitable 
field  for  investigation  along  these  lines. 

ALLERGIC   REACTIONS 

By  the  term  allergic  reaction  in  the  clinical  sense  we  understand 
the  specific  symptomatic  response  on  the  part  of  the  infected  and 
hence  sensitized  organism  to  the  parenteral  reintroduction  of  the  cor- 
responding antigen.  Among  the  infectious  diseases  such  reactions 
have  been  notably  studied  in  connection  with  tuberculosis,  but  are 
evidently  destined  to  play  an  important  role  in  the  diagnosis  of 
other  diseases  as  well.  In  the  present  work  we  shall  confine  our 
attention  to  the  tubercular  test  and  the  luetin  reaction  in  their 
relation  to  the  diagnosis  of  tuberculosis  and  syphilis  respectively. 


ALLERGIC  REACTIONS  311 

The  Tuberculin  Test. — It  will  be  recalled  that  the  tubercular  guinea- 
pig  responds  quite  differently  to  the  introduction  of  living  tubercle 
bacilli  than  does  the  normal  animal.  For  whereas  in  the  latter  a 
local  reaction  occurs  only  after  from  ten  to  fourteen  days,  definite 
changes  can  be  detected  in  the  former  already  within  twenty-four 
to  forty-eight  hours.  But  while  in  the  primarily  non-tubercular 
animal  the  local  lesion  then  remains  active  to  the  end,  local  recovery 
occurs  in  the  reinjected  tubercular  pig.  If  an  emulsion  of  dead 
organisms  (tuberculin)  be  used  instead,  as  much  as  0.5  gram  may 
be  injected,  intraperitoneally  even,  in  the  case  of  the  normal  animal 
without  producing  any  deleterious  results,  while  similar  treatment 
of  the  tubercular  guinea-pig  would  lead  to  a  fatal  ending.  If  the 
injection  is  made  subcutaneously,  and  the  dose  is  chosen  sufficiently 
small  as  not  to  kill,  a  severe  local  reaction  will  result,  as  in  the  first 
instance,  where  living  organisms  were  used,  and  incidentally  it  will  be 
observed  that  in  the  tubercular  in  contradistinction  to  the  non- 
tubercular  animal,  temporarily  at  least,  certain  general  symptoms 
of  illness  develop,  of  which  a  rise  in  temperature  is  the  most  striking 
and  the  most  constant.  Evidently  the  primary  inoculation,  while 
increasing  the  resistance  of  the  animal  to  subsequent  infection  with 
the  organism  in  question  (immunity),  has  called  forth  a  general, 
increased  susceptibility  to  the  action  of  its  products  of  disintegration 
(anaphylaxis) .  According  to  v.  Pirquet  this  difference  in  response 
is  readily  accounted  for,  if  we  remember  that  the  parenteral  intro- 
duction of  foreign  proteins  (in  the  present  instance  of  bacterial  pro- 
teins) leads  to  the  formation  of  corresponding  antibodies,  and  that 
as  a  consequence  of  the  interaction  between  the  two  groups  of  sub- 
stances, in  the  presence  of  complement,  toxic  bodies  (anaphylatoxins) 
are  formed  which  may  then  produce  symptoms  of  variable  nature, 
according  to  the  character  of  the  tissues  which  are  susceptible  to 
their  action. 

In  man  results  have  been  obtained  which  are  perfectly  analogous 
to  those  observed  in  the  guinea-pig.  The  subcutaneous  injection 
of  the  non-tubercular  individual  with  small  doses  of  tuberculin  will 
thus  produce  no  deleterious  consequences  whatever,  while  in  the 
tubercular  subject  the  same  dose  causes  the  well-known  general 
response  by  headache,  muscle  pains,  and  fever,  besides  the  local 
inflammatory  reaction  at  the  site  of  the  injection.  In  cases  where 
the  tubercular  lesion  is  superficially  located  and  can  be  directly 


312  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

observed  the  development  of  increased  redness  and  swelling,  more- 
over, give  evidence  of  a  direct  effect  upon  the  seat  of  infection. 
The  same  is  shown  by  the  increase  in  the  number  of  the  rales  and 
in  the  number  of  bacilli  in  the  sputum,  if  the  injection  is  given  in 
a  case  of  pulmonary  tuberculosis  (focal  reactions). 

If,  on  the  other  hand,  the  tuberculin  is  administered  in  such  a 
manner  that  active  resorption  does  not  occur,  local  reactions  only 
will  be  observed,  and  in  tissues,  it  should  be  remembered,  which 
are  not  tubercular  in  themselves.  Following  a  cutaneous  inoculation 
with  tuberculin  an  inflammatory  papule  thus  appears,  no  matter 
at  what  point  the  injection  is  made  (v.  Pirquet  reaction),  instillation 
into  the  conjunctival  sac  gives  rise  to  an  intense  conjunctivitis 
(Calmette  reaction) ;  innunction  with  a  tubercular  salve  calls  forth  a 
local  dermatitis  (Moro  reaction),  etc. 

Owing  to  this  remarkable  hypersusceptibility  to  tuberculin  on 
the  part  of  the  tubercular  subject,  the  principle  in  question  has  been 
extensively  utilized  for  diagnostic  purposes,  and  it  may  not  be  out 
of  place  to  briefly  describe  the  most  important  methods  which  have 
been  advocated  for  this  purpose. 

The  Tuberculin  Test  According  to  Koch  (Subcutaneous  Method). — 
The  material  which  is  employed  to  this  end  is  the  old  tuberculin  of 
Koch.  Of  this  the  patient  receives  from  0.1  to  1  milligram,  accord- 
ing to  the  condition  of  his  general  health.  In  feeble  individuals 
it  is  best  to  start  with  0.1  milligram,  while  more  robust  persons 
may  take  1  milligram.  The  injections  are  conveniently  given  in 
the  back,  below  the  angle  of  the  scapula,  and  best  during  the  early 
forenoon  hours.  To  wTait  until  the  evening  is  not  advisable,  as  the 
reaction  may  occur  already  after  six  hours  and  might  accordingly 
be  overlooked  during  the  night.  If  no  elevation  of  temperature 
occurs  after  the  first  dose  the  quantity  is  doubled  in  forty-eight  hours, 
and  so  on  until  a  dose  of  10  milligrams,  or  in  individuals  of  feeble 
constitution,  of  5  milligrams  is  reached.  This  Koch  regards  as  the 
limit,  beyond  which  a  reaction  cannot  be  considered  as  specific. 
Should  elevation  of  temperature  follow  any  one  of  the  injections, 
even  though  amounting  to  but  three-tenths  of  a  degree  (C.),  the 
next  dose  should  be  of  the  same  size,  but  it  is  not  to  be  given  until 
the  temperature  has  returned  to  normal.  It  will  often  be  found, 
then,  that  the  second  reaction  is  more  marked  than  the  first.  Such 
an  occurrence  Koch  regards  as  particularly  characteristic,  and, 
indeed,  as  an  infallible  indication  of  the  existence  of  tuberculosis. 


PLATE  X 


Cutaneous  Luetin  Reaction,  Severe.     (Taken 
from  Noguehi.) 


PLATE   XI 


Left 


Right 


Cutaneous    Luetin    Reaction,    Moderate.     (Taken    from    Noguchi.) 


ALLERGIC  REACTIONS  313 

Before  beginning  it  is,  of  course,  desirable  to  observe  the  tempera- 
ture of  the  patient  for  a  while,  and  not  to  inject  until  it  has  been 
found  below  37.3°  C.  for  a  day  or  two.1  The  reaction  is  regarded 
as  positive  if  the  temperature  reaches  a  point  that  is  at  least  0.5°  C. 
above  the  highest  noted  before  the  injection. 

If  small  doses  have  been  used  the  rise  usually  begins  after  ten  to 
sixteen  hours,  while  with  the  larger  doses  it  may  occur  after  six 
to  eight  hours.  There  is  usually  a  slight  chill  which  is  accompanied 
by  headache  and  pains  in  the  muscles,  nausea,  palpitation  of  the 
heart,  etc.  An  hour  or  two  after  the  injection  already  there  may  also 
be  evidence  of  an  inflammatory  reaction  at  the  point  of  inoculation 
(redness  and  tenderness).  At  the  expiration  of  about  ten  hours 
there  is  marked  infiltration  at  this  point  which  may  persist  for  two 
to  six  days  before  resorption  has  taken  place.  After  reaching  its 
highest  point  the  temperature  usually  drops  within  a  few  hours, 
so  that  normal  relations  are  again  restored  at  the  expiration  of 
twenty-four  to  forty-eight  hours  following  the  injection.  The 
patient  may  experience  a  certain  degree  of  lassitude  yet  for  two  or 
three  days  and  possibly  have  an  increased  secretion  of  sputum,  but 
is  then  restored  to  the  same  condition  as  before  the  examination. 

A  positive  reaction,  of  course,  only  means  that  the  patient  has 
a  tubercular  focus  somewhere  in  his  body,  but  does  not  in  itself 
indicate  whether  this  is  active  or  not.  This  point  must  be  decided 
by  the  history,  the  clinical  findings,  etc. 

Regarding  the  constancy  of  the  reaction  in  cases  of  proved  tuber- 
culosis, in  suspected  cases  and  in  supposedly  non-tubercular  individ- 
uals the  accompanying  table  will  furnish  the  desired  information: 

Pulmonary  tuberculosis .       .      .      .     90 . 0  to  100 . 0  per  cent. 

Suspected  cases 92 . 1  per  cent. 

Non-suspected  cases .      .    56 . 1  per  cent. 

• 

The  high  percentage  of  positive  findings  in  non-suspected  cases 
is  readily  explained,  if  we  bear  in  mind  how  common  a  latent,  inac- 
tive tuberculosis  actually  is. 

As  to  indications  and  contra-indications  it  will  suffice  to  state  that 
the  test  may  be  made  in  all  suspected  cases  unless  heart  lesions, 
diabetes,  nephritis,  or  pregnancy  exist,  or  unless  laryngeal  tuber- 
culosis is  suspected. 

1  The  temperature  should  be  taken  every  three  hours. 


314  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

To  illustrate  the  general  safety  of  the  procedure,  providing  that 
the  rules  of  dosage  given  above  are  implicitly  followed,  we  would 
point  out  that  Lowenstein  did  not  meet  with  any  serious  symptoms 
or  a  single  death  in  a  series  of  20,000  single  injections  which  were 
made  under  his  direction. 

The  Tuberculin  Test  According  to  v.  Pirquet  (Cutaneous  Method).— 
The  inner  surface  of  the  forearm  is  cleansed  with  ether,  then  two 
drops  of  the  concentrated  old  tuberculin  of  Koch  are  placed  about 
10  cm.  apart.  With  a  special  instrument,  which  v.  Pirquet  terms 
an  "Impfbohrer"  (vaccination  gimlet),  and  which  is  essentially 
an  exceedingly  fine  chisel  with  a  platinum  iridium  point  that  can 
be  sterilized  in  a  flame,  a  small  abrasion  is  first  produced  midway 
between  the  two  drops.  To  this  end  the  instrument  is  pressed 
against  the  skin  and  rotated,  sufficient  force  being  employed  to 
produce  a  definite  abrasion,  without,  however,  causing  any  bleeding. 
A  similar  scarification  is  then  made  through  each  one  of  the  two  drops 
of  tuberculin.  A  tiny  bit  of  sterile  absorbent  cotton  is  now  laid 
across  each  drop  so  as  to  prevent  it  from  flowing  away.  After  five 
minutes  this  is  removed.  A  dressing  is  not  used.  Should  examina- 
tion at  the  expiration  of  twenty-four  to  forty-eight  hours  not  reveal 
the  existence  of  a  distinct  brown  scab  measuring  about  1  mm.  in 
diameter,  both  at  the  point  of  inoculation  as  well  as  at  that  of  con- 
trol, the  abrasion  has  been  too  slight,  and  the  test  must  be  repeated. 

The  appearance  of  a  positive  reaction  when  fully  developed  is 
well  shown  in  Plate  VII,  and  contrasts  markedly  with  that  of  the 
control.  If  the  abrasions  are  examined  at  frequent  intervals  it  will 
be  observed  that  a  small  wheal  appears  within  a  few  minutes  both 
-at  the  control  and  the  test  point  which  soon  becomes  surrounded 
by  a  pink  halo.  This  disappears  after  a  few  hours,  leaving  a  small 
red  area,  in  the  centre  of  which  a  tiny  scab  begins  to  form.  At 
the  control  point  the  redness  is  still  discernible  after  twenty-four 
hours,  but  then  fades  away.  At  the  test-point,  in  positive  cases, 
the  red  area  begins  to  increase  in  size  after  a  period  of  time  which 
varies  between  three  hours  and  several  days.  Coincidently  the 
inflammatory  area  becomes  elevated  (papular)  and  develops  rapidly 
in  size.  At  the  end  of  forty-eight  hours  the  reaction  has  usually 
reached  its  height.  At  this  time  the  diameter  of  the  "papule"  will 
average  about  10  mm.,  but  it  may  be  much  larger — up  to  30  mm., 
the  size,  cceteris  paribus,  depending  upon  the  quantity  of  tuberculin 


ALLERGIC  REACTIONS  315 

which  has  been  absorbed.  The  centre  of  the  papule  is  sometimes 
pale,  like  an  urticarial  wheal.  The  surface  otherwise  is  frequently 
finely  vesiculated;  pustulation,  however,  never  occurs.  While  ordi- 
narily the  entire  area  is  intensely  hyperemic,  nearly  colorless  papules 
are  sometimes  seen  in  very  advanced  cases  of  tuberculosis,  at  a 
time  when  the  power  of  reaction  on  the  part  of  the  individual  has 
almost  disappeared  (cachectic  reactions).  The  hyperemic  area  is 
usually  limited  to  the  papule  itself,  but  occasionally  extends  beyond, 
forming  an  areola,  which  strongly  reminds  one  of  what  is  seen  in 
cases  of  vaccination. 

After  having  reached  its  height  the  exudation  gradually  subsides. 
The  swelling  disappears  in  from  five  to  eight  days,  but  the  pigmen- 
tation which  then  develops  frequently  remains  visible  for  a  number 
of  weeks. 

Exceptionally  the  reaction  does  not  begin  to  develop  until  after 
twenty-four  hours  following  the  inoculation.  Such  a  delayed 
response  v.  Pirquet  speaks  of  as  a  torpid  reaction.  This  is  notably 
seen  in  individuals  who  show  no  clinical  evidence  of  tuberculosis, 
and  is  the  more  frequent  the  older  the  patient. 

The  great  advantage  of  v.  Pirquet 's  method  as  compared  with  the 
older  subcutaneous  method  of  Koch  is,  of  course,  its  simplicity,  and 
the  fact  that  undesirable  systemic  effects  hardly  ever  occur,  provid- 
ing that  the  abrasion  has  been  made  lege  artis,  and  that  opportunity 
for  undue  absorption  has  not  been  afforded.  As  in  the  case  of  the 
subcutaneous  method,  however,  a  positive  reaction  merely  denotes 
the  presence  of  a  tubercular  focus  somewhere  in  the  body,  which 
need  not  be  active,  however,  and  the  diagnostic  value  of  the  method 
is  hence  limited  to  the  same  extent  and  even  more.  The  greatest 
sphere  of  usefulness  indeed  seems  to  lie  in  its  application  to  the 
diagnosis  of  tuberculosis  in  very  young  children.  From  a  study 
of  757  children  in  which  the  test  had  been  applied  by  v.  Pirquet 
and  in  which  the  results  were  compared  with  the  clinical  findings, 
it  appears  that  of  the  clinically  tubercular  cases  87  per  cent, 
gave  the  reaction,  while  this  was  also  found  in  20  per  cent,  of 
the  non-suspected  cases.  The  negatively  reacting  tubercular  cases, 
v.  Pirquet  points  out,  were  almost  exclusively  cachectic  or  in  the 
last  stages  of  miliary  tuberculosis. 

As  the  result  of  a  study  of  124  children  which  had  come  to  autopsy 
and  in  which  the  test  had  been  made,  v.  Pirquet  concludes  that  a 


316  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

positive  cutaneous  reaction  is  never  observed  in  the  absence  of  a 
tubercular  lesion;  that  a  negative  reaction  ordinarily  indicates  freedom 
from  tuberculosis,  but  that  such  a  result  may  also  be  obtained  in 
the  last  stages  of  the  disease.  As  a  positive  reaction  may  be  expected 
in  over  90  per  cent,  of  all  individuals  after  the  fourteenth  year,  it  is 
clear,  however,  that  the  diagnostic  significance  of  the  reaction  is 
then  practically  nil.  As  35  per  cent,  of  all  children,  moreover,  give 
a  positive  cutaneous  reaction  between  the  ages  of  six  and  ten  already, 
it  is  evident  that  even  at  this  age  its  diagnostic  value  is  limited. 

The  Tuberculin  Test  According  to  Calmette  (Conjunctival  Method). 
—While  Calmette  advocates  the  use  of  a  tuberculin  which  essentially 
contains  the  alcohol-insoluble  constituents  of  bovine  tubercle  bacilli, 
made  up  into  a  J  per  cent,  aqueous  solution,  one  may  also  employ 
a  5  per  cent,  solution  of  the  old  tuberculin  of  Koch.  One  or  two 
drops  of  either  solution  are  placed  upon  the  conjunctiva  of  one  eye 
near  its  inner  canthus,  when  the  lids  are  held  together  for  about  a 
minute.  In  the  normal  individual  slight  redness  may  then  develop 
and  persist  for  a  few  hours,  after  which  it  disappears.  In  the  tuber- 
cular subject,  on  the  other  hand,  marked  hyperemia  occurs  after 
three  to  six  hours  (more  rarely  after  twelve  to  twenty-four  hours) ; 
this  principally  affects  the  lower  lid,  the  lower  portion  of  the  eyeball, 
the  caruncle  and  the  semilunar  fold  (see  Plate  VIII).  At  the  same 
time  there  is  some  swelling  and  secretion,  which  in  severe  reactions 
becomes  mucopurulent. 

The  height  of  the  reaction  is  reached  after  ten  to  twelve  hours, 
after  which  the  inflammatory  manifestations  usually  disappear  and 
there  is  a  return  to  the  normal. 

While  in  most  cases  no  unduly  severe  reactions  occur,  such  have 
nevertheless  been  noted  in  isolated  cases,  and  a  number  of  observers 
look  upon  the  method  in  its  original  form  as  dangerous  and  not 
justifiable.  Eppenstein  accordingly  recommends  successive  tests 
with  solutions  of  increasing  strength,  and  the  use  of  both  eyes 
alternately,  beginning  in  adults  with  a  1  per  cent,  solution  of  the 
old  tuberculin,  and  then  increasing  to  a  2  per  cent,  and  finally 
to  a  4  per  cent,  solution,  while  in  children  a  \  per  cent,  solution 
is  used  as  the  starting  dose. 

The  existence  of  any  disease  of  the  eye  would,  of  course,  constitute 
a  contra-indication  to  the  method  in  question. 

As  regards  the  clinical  value  of  the  Calmette  reaction,  as  com- 


THE  LUETIN  REACTION  317 

pared  with  the  cutaneous  reaction  of  v.  Pirquet,  it  appears  from  an 
analysis  of  2974  examinations  collected  by  Petit  that  94.3  per  cent, 
of  clinically  tubercular  cases  showed  the  reaction,  while  among  non- 
tubercular  individuals  only  18.4  per  cent,  reacted.  The  eye  reaction 
would  thus  seem  to  be  more  useful  from  the  diagnostic  standpoint, 
and  it  is  to  be  hoped  that  it  may  yet  be  improved  to  such  a  degree 
that  dangerous  reactions  may  with  certainty  be  avoided. 

As  in  the  case  of  the  v.  Pirquet  reaction,  systemic  and  focal 
symptoms  do  no  occur. 

The  Tuberculin  Test  According  to  Moro  (Dermo-reaction). — Moro 
has  shown  that  a  skin  reaction  may  be  obtained  in  tubercular  individ- 
uals after  inunction  with  a  salve  composed  of  equal  parts  of  the 
old  tuberculin  of  Koch  and  of  lanolin.  To  this  end  a  small  amount 
of  the  salve  (about  the  size  of  a  pea)  is  for  a  minute  rubbed  into  an 
area  of  the  skin  measuring  not  more  than  5  cm.  in  diameter.  The 
best  district  for  this  purpose  is  the  skin  just  below  the  sternum  or 
in  the  vicinity  of  the  nipple.  After  drying  for  about  ten  minutes 
the  patient  may  dress,  no  special  covering  being  required.  After 
twenty-four  to  forty-eight  hours  a  dermatitis  then  develops  which 
is  characterized  by  the  appearance  of  miliary  nodules  of  variable 
size  and  number,  which  occur  either  singly  or  confluent.  At  the 
same  time  there  is  a  more  or  less  extensive  general  redness  of  the 
affected  area,  accompanied  by  a  certain  amount  of  itching  (see 
Plate  IX). 

Regarding  the  clinical  value  of  the  method  our  knowledge  is  as 
yet  too  meager  to  warrant  its  general  recommendation,  v.  Pirquet 
states  that  he  has  been  able  to  obtain  positive  results  only  in  highly 
susceptible  individuals,  but  suggests  that  it  may  be  tried,  if  for 
any  reason  the  cutaneous  or  the  eye  reaction  cannot  be  employed. 


THE   LUETIN   REACTION 

While  a  number  of  different  investigators  had  previously  attempted 
a  skin  reaction  diagnosis  in  connection  with  syphilis,  satisfactory 
results  could  hardly  be  expected  so  long  as  the  successful  cultivation 
of  the  corresponding  spirochete  in  pure  culture  had  not  been  accom- 
plished. The  solution  of  the  latter  problem  we  owe  to  the  painstaking 
work  of  Noguchi,  and  to  the  same  investigator  belongs  the  credit 


318  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

of  having  first  prepared  an  antigen  with  which  a  specific  syphilitic 
reaction  may  be  obtained  in  a  large  percentage  of  infected  individuals. 

Preparation  of  the  Antigen. — Pure  placental  ascites  agar  cultures 
of  the  pallida  are  ground  up  in  a  mortar  with  placental  ascites 
bouillon  cultures  of  the  organisms  until  a  fairly  thin  emulsion  is 
obtained.  This  is  sterilized  for  one  hour  at  60°  C.  and  treated  with 
tricresol  to  the  extent  of  0.5  per  cent.  The  resultant  product  Noguchi 
has  termed  luetin.  After  being  tested  for  its  sterility  it  is  ready 
for  use.  A  similar  preparation  is  made  from  sterile  culture  material 
and  constitutes  the  control  fluid. 

Injection  of  the  Patient. — Before  using,  the  contents  of  the  luetin 
bottle,  which  must  be  kept  in  the  ice-box  and  frequently  examined 
for  its  sterility,  should  be  thoroughly  shaken,  so  as  to  bring  about 
an  even  suspension  of  the  spirochetes.  With  a  sterile  pipette  equal 
parts  of  the  luetin  and  sterile  saline  are  then  placed  in  a  tuberculin 
syringe  and  0.07  c.c.  injected  in  the  case  of  an  adult  and  0.05  c.c. 
in  infants.  The  injections  are  made  intracutaneously ,  the  left  arm 
being  chosen  for  the  luetin  and  the  right  arm  for  the  control  fluid, 
of  which  a  corresponding  amount,  likewise  diluted  with  saline,  is 
used.  Separate  syringes  are  kept  for  the  luetin  and  the  control 
fluid.  Noguchi,  moreover,  advises  that  each  arm  be  injected  at 
two  points,  about  5  cm.  apart.  Considering  the  severity  of  some  of 
the  reactions,  however,  I  should  personally  advise  single  injections. 

Reactions. — While  in  non-syphilitic  individuals  the  effect  of  the 
luetin  and  the  control  injection  is  identical  and  merely  represents 
a  slight  traumatic  reaction,  which  recedes  within  forty-eight  hours 
and  leaves  no  induration,  there  may  be  a  marked  difference  between 
the  two  sides  in  syphilitic  persons.  Noguchi  here  distinguishes  three 
types  of  reaction  at  the  points  where  the  luetin  has  been  injected. 
In  the  first  or  papular  form  "a  large,  raised,  reddish,  indurated 
papule,  usually  from  5  to  10  mm.  in  diameter,  makes  its  appearance 
in  twenty-four  to  forty-eight  hours.  The  papule  may  be  surrounded 
by  a  diffuse  zone  of  redness  and  show  marked  telangiectasis.  The 
dimensions  and  the  degree  of  induration  slowly  increase  during  the 
following  three  or  four  days,  after  which  the  inflammatory  processes 
begin  to  recede.  The  color  of  the  papule  gradually  becomes  dark 
bluish  red.  The  induration  disappears  within  one  week,  except  in 
certain  instances,  in  which  a  trace  of  the  reaction  may  persist  for  a 
longer  period.  This  latter  effect  is  usually  seen  among  patients  with 


THE  LUETIN  REACTION  319 

secondary  syphilis  under  regular  mercurial  treatment  in  whom  there 
are  no  manifest  lesions  at  the  time  of  making  the  skin  test.  Patients 
with  congenital  syphilis  also  show  this  reaction  in  the  early  period  of 
life."  In  the  second  pustular  form  "the  beginning  and  course  of  the 
reaction  resemble  the  papular  form  until  about  the  fourth  day, 
when  the  inflammatory  processes  commence  to  progress.  The 
surface  of  the  indurated,  round  papule  becomes  mildly  edematous, 
and  multiple  miliary  vesicles  occasionally  form.  At  the  same  time 
a  beginning  central  softening  of  the  papule  can  be  seen.  Within 
the  next  twenty-four  hours  the  papule  changes  into  a  vesicle,  filled 
at  first  with  a  semi-opaque  serum  that  later  becomes  definitely 
purulent.  Soon  after  this  the  pustule  ruptures  spontaneously  or 
after  slight  friction  or  pressure.  The  margin  of  the  broken  pustule 
remains  indurated,  while  the  defect  caused  by  the  escape  of  the 
pustular  contents  becomes  quickly  covered  by  a  crust  that  falls  off 
within  a  few  days.  About  this  time  the  induration  usually  disappears, 
leaving  almost  no  scar  after  healing.  There  is  a  wide  range  of 
variation  in  the  degree  of  intensity  of  the  reaction  described  in 
different  cases,  as  some  show  rather  small  pustules,  while  in  others 
the  pustule  is  much  larger.  This  reaction  was  found  almost  constant 
in  patients  with  tertiary  or  late  hereditary  syphilis"  (see  Plate  X). 

In  the  third  or  torpid  form,  which  was  only  noted  in  rare  instances, 
"the  injection  sites  fade  away  to  almost  invisible  points  within 
three  or  four  days,  so  that  they  may  be  passed  over  as  negative 
reactions.  But  sometimes  these  spots  suddenly  light  up  again  after 
ten  days,  or  even  longer,  and  progress  to  small  pustular  formation. 
The  course  of  this  pustule  is  similar  to  that  described  for  the  preceding 
form. 

"This  form  of  reaction  has  been  observed  in  a  case  of  primary 
syphilis,  in  one  of  hereditary  syphilis,  and  in  two  cases  of  secondary 
syphilis,  all  being  under  mercurial  treatment. 

"If  no  change  occurs  after  four  or  five  days  the  reaction  should 
accordingly  not  be  termed  negative  and  Noguchi  advises  that  the 
observation  of  the  patient  be  continued  for  at  least  three  or  four 
weeks. 

"Neither  in  syphilitics  nor  in  parasyphilitics  did  a  marked  consti- 
tutional effect  follow  the  intradermic  inoculation  of  the  luetin.  In 
most  positive  cases  a  slight  rise  in  temperature  took  place,  lasting 


320  IMMUNOLOGICAL  METHODS  OF  DIAGNOSIS 

for  one  day.  In  three  tertiary  cases  and  in  one  hereditary  case, 
however,  general  malaise,  loss  of  appetite,  and  diarrhea  were  noted." 

Results. — As  regards  the  specificity  and  the  value  of  the  Noguchi 
reaction  from  a  diagnostic  standpoint  there  can  be  but  little  doubt, 
and  it  seems  from  the  data  which  are  thus  far  available  that  it  is 
especially  serviceable  in  the  late  stages  of  the  disease,  and  in  the 
recognition  of  congenital  cases. 

Noguchi  expresses  the  belief  that  the  allergic  condition  of  the 
skin  persists  as  long  as  the  infecting  agent  still  survives  somewhere 
in  the  body,  and  that  its  disappearance,  cceteris  paribus,  implies  the 
cure  of  the  patient.  It  is  to  be  noted,  however,  that  cases  occur  in 
which  the  disease  persists  in  spite  of  treatment  and  in  spite  of  the 
absence  of  the  luetin  reaction. 

Kammerer,  who  has  recently  repeated  Noguchi's  work,  sums  up 
his  experiences  as  follows: 

The  intracutaneous  reaction  is  devoid  of  danger  and  entails  no 
special  discomfort  for  the  patient.  Aside  from  one  uncertain  case 
it  was  specific  for  syphilis.  A  differentiation  between  the  specific 
and  non-specific  traumatic  reactions  is  possible  in  most  though 
not  in  all  cases.  In  cases  of  marked  reaction  the  control  site  also 
often  responds  to  the  point  of  vesicle  or  even  pustule  formation. 
Of  the  cases  examined  which  w^ere  known  to  be  syphilitic  more  than 
half  did  not  give  the  reaction,  the  highest  percentage  of  positive 
findings  occurring  in  late  cases.  In  view  of  the  occurrence  of  retarded 
reactions  the  patients  should  be  observed  for  two  weeks. 

My  own  observations  have  been  rather  scanty.  In  the  beginning, 
when  0.5  per  cent,  carbolic  was  used  as  a  preservative,  it  was  difficult 
to  guard  the  preparation  against  contamination.  With  the  use  of 
the  cresol  the  danger  from  this  source  is  certainly  less,  but  I  must 
confess  that  a  couple  of  very  severe  local  reactions  (very  curiously 
most  severe  on  the  control  side)  have  somewhat  dampened  my 
enthusiasm  regarding  the  general  applicability  of  the  test.  Further 
experience  with  it  will  be  necessary  before  it  can  be  recommended 
for  routine  use. 

As  regards  the  comparative  value  of  the  Wassermann  and  the 
luetin  reaction  it  is  still  too  early  to  make  any  definite  statements. 

For  the  present  it  will  no  doubt  be  advisable  to  control  the  one 
bv  the  other. 


INDEX 


A 


^BDERHALDEN'S  pregnancy  test,  307 
Actinogestin,  146 
Agglutination  reactions,  282 
Agglutinins,  95 

partial,  112 
Aggressins,  35,  42 

artificial,  44 

natural,  44 
Aggressivity,  active,  32,  41 

of  parasites,  32 

differences  in,  34 

passive,  35 
Albuminolysins,  103 
Aleppo  boil,  salvarsan  in,  276 
Alexins,  69,  99 

leukocytic,  79 
Allergens,  92,  104,  150 
Allergia,  91,  98,  104 
Allergic  diagnostic  reactions,  310 
luetin  test,  317 
tuberculin  test,  311 

reactions,  159 
Allergin,  150 
Amboceptors,  69,  99,  119 

chemical  nature  of,  77 

immune,  90 

mode  of  action  of,  70 

normal,  69 

origin  of,  76 

specificity  of,  74 

Amebiasis,  use  of  salvarsan  in,  276 
Anaphylactic  shock,  104,  151 

mechanism  of,  151,  155 

toxin,  152 

Anaphylactin,  104,  150 
Anaphylactogens,  150 
Anaphylatoxin,  152 
Anaphylaxis,  104,  144,  147 

and  idiosyncrasies,  167 

passive,  150,  151 

relation  of,  to  diseases,  158 
Animal  passage,  effect  of,  on  virulence, 

36 

Animalized  bacteria,  36 
Anthrax,    sympathetic,    vaccination 

against,  200 

Antiaggressin  immunity,  42,  135 
Antiaggressins,  42 
Antiamboceptors,  101 
21 


Antianaphylaxis,  149,  156 

mechanism  of,  156 
Antibacterial  immunity,  137 
Antibodies,  89,  92 
formation  of,  114 
interaction  between,  and  antigens, 

108 

Antiferments,  102 
Antigens,  89,  92,  108 
Antilipoids,  103 
Antimeningococcus  serum,  242 

administration  of,  244 

dosage  of,  244 

preparation  of,  243 

results  of  treatment  with,  245 

standardization  of,  243 
antiricin,  106 
Antistreptococcus  serum,  246 

dosage  of,  249 

preparation  of,  248 

results  of  treatment  with,  251 

standardization  of,  248 

uses  of,  249 

Antitoxic  immunity,  132 
immunization,  223 
treatment  of  cholera,  240 

of  diphtheria,  223 

of  dysentery,  238 

of  plague,  241 

of  tetanus,  234 

Antitoxin,  action  of,  upon  toxin,  106 
Antitoxins,  92 
Antitrypsin,  102 
Antivenin,  106 
Arthus'  phenomenon,  147 
Asthmatic  idiosyncrasies,  168 
Athrepsia,  135 
Athreptic  immunity,  134 
Attenuation  of  virulence,  40 
Auto-antibodies,  100 
Autocytotoxins,  101 
Autoserum  therapy,  255 

in  cancer,  255 

in  syphilis,  256 


B 


BACTERIAL  emulsions,  preparation  of, 

201 
poisons,  47 


322 


INDEX 


Bacterial  proteins,  49,  52 
Bactericidal  immunity,  137 
substances  of  blood,  71 

demonstration  of,  71 
estimation  of,  72 
Bacteriolysins,  94,  98 
Bacteriolytic-bacteriotropic    immuniza- 
tion, 241 
against  gonococcus  infections, 

254 

against   meningococcus   infec- 
tions, 241 

against    pneumococcus    infec- 
tions, 252 
against  staphylococcus  infec- 

tions,  254 

against    streptococcus     infec- 
tions, 246 

Bacteriolytic  diagnostic  reactions,  288 
Bacteriotropins,  61,  101 
Bilharziasis,  use  of  salvarsan  in,  276 
Biological  blood  test,  304 
Blood  test,  biological,  304 
Buckwheat  poisoning,  170 


CALMETTE'S  tuberculin  test,  316 
Cancer,  chemotherapy  in,  278 
Capsule  bacteria,  35 

formation,  factors  determining,  35, 

37 

Cattle  plague,  vaccination  against,  200 
Chemoreceptors,  115,  261 
Chemotaxis,  65 

negative,  65 

positive,  65 
Chemotherapy,  259 

in  bacterial  infections,  277 

in  cancer,  278 

in  protozoon  infections,  259,  276 
Cholera,  antitoxic  treatment  of,  240 

preparation  of  vaccine  for,  196 

results  following  use  of  vaccine,  197 

serum  diagnosis  of,  288,  289 

vaccination  against,  195 
Complement,  69,  76,  99 

chemical  nature  of,  77 

fixation,  diagnostic  reactions  based 
upon,  290 

mode  of  action  of,  70 

origin  of,  76,  78 

structure  of,  76 
Complementoid,  77 
Complementophilic  group,  119 
Cytase,  69 
Cytolysins,  97 
Cytotoxins,  98 


Diphtheria,      antitoxic      immunization 

against,  223 
antitoxin,  223 

contra-indicationsto  use  of,  230 
dosage  of,  228 
preparation  of,  224 
results  of  treatment  with,  232 
titration  of,  226 
uses  of,  228 
Drug  fastness,  262 

idiosyncrasies,  170 

Dysentery,  antitoxic  treatment  of,  238 
antitoxin,  238 

dosage  of,  239 
preparation  of,  238 
results  of  treatment  with,  239 
uses  of,  239 

prophylactic    treatment    of,    with 
vaccine,  200 


E 


EHRLICH'S  side-chain  theory,  106 
Eisenberg  phenomenon,  112 
Endolysins,  79 

leukocytic,  79 
Endotoxins,  49,  52 
Epitheliolysins,  98 
Ergins,  104 
Ergophpric  group,  119 
Exotoxins,  49 


FAGOPYRISMUS,  170 

Ferment  reactions,  307 

Fixateur,  69 

Flexner  serum.    See  Antimeningococcus 

serum,  242 
Frambesia,  use  of  salvarsan  in,  276 


GONOCOCCUS  infections,  serum  diagnosis 

of,  303 

treatment  of,  254 
Grease,  177 


HAPTINS,  118 
Haptophoric  group,  116 
Hay  fever,  168 
Hemolysins,  98 
Hepatolysins,  98 
Heterolysins,  100 


DEFENSIVE    forces    of    macroorganism,    IDIOSYNCRASIES  and  anaphylaxis,  167 
57  Immune  body,  99 


INDEX 


323 


Immune  opsonins,  101 

sera,  90 

Immunological  diagnostic  methods,  282 
Immunology,  19 
Immunity,  absolute,  129 
acquired,  131 
active,  89,  132,  140 
antiaggressin,  42,  135 
antibacterial,  132,  137 
antiblastic,  39 
antitoxic,  132 
artificial,  131 
athreptic,  134 
class,  129 
definition  of,  127 
generic,  130 
histogenetic,  140 
mechanism  of   different   types  of, 

133 

natural,  127 
passive,  89,  133,  140 
racial,  130 
species,  130 
types  of,  127 
Immunization,  172 
active,  172 

against  cholera,  195 
against  dysentery,  200 
against  plague,  197 
against  rabies,  183 
against  smallpox,  175 
against  typhoid  fever,  189 
for  prophylactic  purposes,  172 
for  therapeutic  purposes,  200 
in  pyogenic  infections,  201 
in  tuberculosis,  206 
antitoxic,  223 

against  cholera,  240 
against  diphtheria,  223 
against  dysentery,  238 
against  plague,  241 
against  tetanus,  234 
bacteriolytic-bacteriotropic,  241 
against  gonococcus  infections, 

254 

against  meningitis,  241 
against    pneumococcus    infec- 
tions, 252 

against   staphylococcus   infec- 
tions, 254 

against     streptococcus     infec- 
tions, 246 
passive,  222 
Infection,  20,  21,  47 

local  conditions  favoring,  23 
nature  of,  23 
obstacles  to,  25 
with  animal  parasites,  55 
with  necro-parasites,  29 
with  semiparasites,  31 
with  true  parasites,  30 
Infectious  disease,  20,  21,  47 
Infectiousness,  32 


Intermediary  body,  69 
Isocytolysins,  100 
Isolysins,  100 


KALA-AZAR,  use  of  salvarsan  in,  276 
Koch's  tuberculin,  207,  208,  209 

test,  312 
Kretz,  paradox  of,  145 


LEISTUNGSKERN,  114 
Leukins,  79 

Leukocytes,  washed,  215 
Leukolysins,  98 
Luetin,  preparation  of,  318 

reaction  of  Noguchi,  317 

use  of,  318 


M 


MACROPHAGES,  58 

Malaria,  use  of  salvarsan  in,  276 

Malignant  disease,  chemotherapy  in, 
278 

Meningococcus  meningitis,  serum  treat- 
ment of,  241 

Microphages,  58 

Moro's  tuberculin  test,  317 


N 


NECROPARASITES,  29 

offensive-defensive  reaction  in  in- 
fections with,  80 
Negative  phase  of  Wright,  220 
Neosalvarsan,  268 
Nephrolysins,  98 
Neurolysins,  98 
Neurorelapses  in  syphilis  following  use 

of  salvarsan,  272 
Noguchi's  antigen,  292 

luetin  reaction,  317 
Normal  serum  therapy,  257 
Nutriceptors,  116,  141,  261 


OPSONIC  content  of  blood,  67 

estimation  of,  214 

index,  218 

Opsonification  of  bacteria,  62,  64 
Opsonins,  immune,  101 

normal,  61 
Organotropism,  260 


324 


INDEX 


PARADOX  of  Kretz,  145 
Parasites,  necro-,  29 

semi-,  31 

tissue,  32 

transitional,  31 

true,  30 

offensive-defensive  reaction  in 

infections  with,  81 
Parasitotropism,  260 
Pfaundler's  thread  reaction,  96 
Pfeiffer's  phenomenon,  71,  94 

test  for  cholera,  288,  289 
Phagocytic  function  of  cells,  58 

index,  218 
Phagocytosis,  57 

Phenomenon  of  Theobald  Smith,  148 
Pirquet's  tuberculin  test,  314 
Plague,  antitoxic  treatment  of,  241 

preparation  of  vaccine,  198 

results  following  the  use  of  vaccine, 
197 

vaccination  against,  197 
Pneumococcus  infections,  chemotherapy 

in,  277 

serum  treatment  of,  252 
Poisons,  bacterial,  47 
Pollen  disease,  168 
Positive  phase  of  Wright,  220 
Precipitin  reactions,  304 
Precipitinogens,  97,  304 

partial,  304 
Precipitins,  96 

Pregnancy,  serum  diagnosis  of,  307 
Proteins,  bacterial,  49,  52 
Ptomains,  48 


RABIES,  preparation  of  virus  for  pre- 
ventive treatment,  184 
results  of  preventive  vaccination, 

188 

vaccination  against,  183 
Receptoric  atrophy,  immunity  due  to, 

141,  144 
Receptors,  115 

classification  of,  118 
formation  of,  121 
structure  of,  118 

Relapsing  fever,  use  of  salvarsan  in,  276 
Retro  vaccination  lymph,  178 


S 


SALVARSAN,  265 

centra-indications  to  use  of,  271 
dosage,  269 

frequency  of  injection,  269 
method  of  treatment  with,  266 


Salvarsan,  neurorelapses,  following  use 

of,  in  syphilis,  272 
reactions  following  use  of,  268 
results    of    treatment    of    syphilis 

with,  273 

uses  in  non-syphilitic  maladies,  276 
Semiparasites,  31 

offensive-defensive  reaction  in  in- 
fections with,  85 
Sensibilisin,  150 
Sensibilisinogen,  150 
Serum  sickness,  150 

therapy,  normal,  257 

immune,  223 

Side-chain  theory  of  Ehrlich,  106 
Side  chains,  114 
Sleeping  sickness,  use  of  salvarsan  in, 

276 
Smallpox,  preparation  of  vaccine  against, 

178 

vaccination  against,  175,  180 
Smith,  Theobald,  phenomenon  of,  103, 

148 

Spermatolysins,  98 

Staphylococcus  infections,  serum  treat- 
ment of,  254 

Streptococcus  infections,   serum  treat- 
ment of,  246 

Substance  sensibilisatrice,  69,  99 
Swine  plague,  vaccination  against,  200 
Syphilis,  autoserum  therapy  in,  256 
diagnosis  of,  by  luetin  reaction,  294 

by  Wassermann  reaction,  271 
neurorelapses  in,  following  use  of 

salvarsan,  272 
treatment   of,    with   neosalvarsan, 

268 
with  salvarsan,  265 


TETANOLYSIN,  106 

Tetanus,  antitoxic  treatment  of,  234 

antitoxin,  234 

dosage  of,  235 

results  of  treatment  with,  236 
uses  of,  235 

Therapia  magna  sterilisans,  143,  264 
Tissue  parasites,  32 
Torrey's  serum,  254 
Toxin,  anaphylactic,  152 
Toxin-antitoxin,  interaction,  106 
Toxins,  true,  49 
Toxogenin,  151 
Toxoid,  120 
Toxons,  123 
Toxophoric  group,  116 
Transitional  parasites,  31 
Tuberculin,  207 

indications  and  contra-indications 
to  use  of,  211 

Koch's  method  of  using,  208 


INDEX 


325 


Tuberculin,  new,  20S,  209 
old,  207,  208 

reactions  following  use  of,  211 
test  of  Calmette,-  316 
of  Koch,  312 
of  Moro,  317 
of  v.  Pirquet,  314 
T.  O.,  208 
T.  R.,  208 

Wolff-Eisner's  method  of  using,  210 
Wright's  method  of  using,  210 
Tuberculosis,  vaccine  treatment  of,  206 
Tumor  immunity,  135 
Typhoid  fever,  antitoxic  treatment  of, 

241 

preparation  of  vaccine,  190 
prophylactic  treatment  of,  189 
results  following   vaccination, 

194 

vaccination  against,  189 
Typhus  fever,  use  of  salvarsan  in,  276 


UNICEPTORS,  119 
Urticarial  idiosyncrasies,  169 


VACCINATION,  175 

against  cholera,  195 

against  dysentery,  200 

against  plague,  197 

against  rabies,  183 

against  smallpox,  177,  180 

against  typhoid  fever,  189 

curative    value    of,     in    pyogenic 

infections,  201 
in  tuberculosis,  206 
protective  value  of,  against  cholera, 

195 

against  dysentery,  200 
against  plague,  197 


Vaccination,  protective  value  of,  against 

rabies,  188 

against  smallpox,  177,  182 
against  typhoid  fever,  194 
in  pyogenic  infections,  201 
in  tuberculosis,  206 
Vaccine,    preparation    of    anti-cholera, 

196 

of  anti-plague,  198 
of  anti-rabies,  184 
of  anti-smallpox,  178 
of  anti-typhoid,  189 
of  bacterial,  201 
of  tuberculin,  207,  208 
smallpox,  preparation  of,  178 
Vaccines,  autogenous,  201 
bacterial,  201 

standard  doses  of,  204 
standardization  of,  190 
indications  for  use  of,  205 
polyvalent,  202 
stock,  201 

Vaccine  treatment.    See  Vaccination. 
Variolation,  176 
Vincent's  angina,  use  of  salvarsan  in, 

276 

Virulence,  32,  33 
actual,  37 
attenuation  of.  40 
organ,  39 
potential,  37 
Virus  fixe,  183 
street,  183 


W 


WASSERMANN  reaction,  103,  291 
diagnostic  value  of,  299 
provocative,  274,  300 
technique,  292 

Widal  reaction,  95,  283,  284 

macroscopic  method,  286 
microscopic  method,  284 


Si. 


.. 


m 


^^m 


r 


^^^m 


